Almost nothing is known of Knox's life before 1540, the accounts given by his earlier biographers being mostly fanciful. Of his parentage it is known only that his mother's name was Sinclair (Knox used the name John Sinclair as an incognito in times of danger), that his father's name was William, and that he and both Knox's grandfathers had fought, and two of them had died—perhaps at the Battle of Flodden against Henry VIII's troops. The family may have been farmers.

It is supposed that Knox trained for the priesthood under the scholar John Major, most probably at the University of St. Andrews. Knox did not take a master's degree, however, but he ended his training with a mind imbued with that delight in abstract thought and dialectical disputation which, even in that age, was recognized throughout Europe as typical of Scottish scholarship. He was in priest's orders by 1540, and in 1543 he was known to be also practicing as an apostolic notary in the Haddington area, which would seem to indicate that he was in good standing with the ecclesiastical authorities.

Two years later, however, Knox was in more equivocal company as tutor to the sons of two gentlemen of East Lothian who were deeply involved in the intrigues of political Protestantism. Under their protection, George Wishart (Wishart, George), a Scottish Reformation leader who was to become an early martyr for the cause, began a preaching tour in the Lothians in December 1545. Knox was much in his company, and Knox's complete conversion to the Reformed faith dates from his contact with Wishart, whose memory he cherished ever afterward. Wishart was burned for heresy in March 1546 by Cardinal David Beaton (Beaton, David), archbishop of St. Andrews, who, rather than the weak governor, was the real ruler of Scotland. Wishart's execution began a chain of events that profoundly altered Knox's life. Three months later, Beaton was murdered by Protestant conspirators who fortified themselves in St. Andrews castle.

Meantime, Knox, accompanied by his pupils, was moving from place to place to escape persecution and arrest. His desire was to go to Germany to study there at the Protestant seats of learning, but his employers sent word to him to take their sons to St. Andrews (Saint Andrews) and continue their education under the protection of the castle. Thus, in April 1547, less than a year after the cardinal's murder and against his own desire, Knox arrived with his pupils in St. Andrews—still an unknown man. The three months that he spent there transformed him, against his own predisposition, into the acknowledged spokesman and protagonist of the Reformation movement in Scotland. The Protestants in the castle had become involved in controversy with the university; several of them, becoming aware that a man of uncommon gifts had joined them, pressed upon Knox's conscience the duty of taking up “the public office and charge of preaching.” Knox's inclination was for the quiet of the study and the schoolroom, not for the responsibilities and perils of the life of a preacher of a proscribed and persecuted faith. He resisted the call with tears, and only after great hesitation was he persuaded to preach in the town of St. Andrews a sermon that convinced friend and foe alike that the great spokesman of Scottish Protestantism had been found. This was the turning point of Knox's life; from this time forward he regarded himself as called to preaching by God, and he was the more certain of the divine origin and compulsion of the call in that it ran counter to every inclination of his own.

At the end of June 1547, French assistance reached the governor of Scotland. The garrison of St. Andrews castle, bombarded from without and assailed by plague within, capitulated on terms that were not kept; Knox and others were carried off to slavery in the French galleys. English intervention secured his release 19 months later, though with permanently broken health.

In England the Protestant government of Edward VI was endeavouring to hurry clergy and people into the Reformation faster, if anything, than most of them were willing to go. For this program preachers and propagandists were urgently required; and because a return to a Scotland under Roman Catholic rule was impossible for Knox at this time, the English government promptly made him one of a select corps of licensed preachers and sent him north to propagate the Reformation in the turbulent garrison town of Berwick-upon-Tweed. He brought order to the town and established a congregation on Puritan lines, and there he met Marjorie Bowes, who was to become his wife. Early in 1551 he was given a new assignment in Newcastle and a little later was appointed to be one of the six royal chaplains whose duties included periodic residence at, and preaching before, the court as well as itinerant evangelism in areas where the regular clergy were lacking in Protestant zeal. He later refused to accept the bishopric of Rochester and the vicarage of Allhallows, London, but continued, under the patronage of the government, to exercise an itinerant ministry, mainly, but not exclusively, in Buckinghamshire, Kent, and London.

In three respects Knox left his mark on the Church of England (England, Church of): he took part in the shaping of its articles; he secured the insertion into The Book of Common Prayer (Book of Common Prayer) of the so-called black rubric, which denies the corporal presence of Christ in the consecrated bread and wine used in Holy Communion and explains that kneeling at communion implies no adoration of the elements; and he was one of the chief foster fathers of English Puritanism, a reform movement started within the state church with a view to the more rigorous application of Reformation principles in doctrine and worship.

On the accession of Mary Tudor (Mary I), a Roman Catholic, to the throne in 1553, Knox was one of the last of the Protestant leaders to flee the country. He escaped to the Continent disturbed by the realization that the fate of “true religion” in England had turned on the religious opinions of one woman. He could see no security for the Reformation anywhere if the personal whim of a sovereign was permitted to settle the religion of a nation. Might it not be legitimate for Protestant subjects, in such circumstances, to resist—if necessary by force—the subversion of their religion by a Roman Catholic ruler? Knox formulated his fateful conclusion, later to be applied in Scotland, that God-fearing magistrates and nobility have both the right and the duty to resist, if necessary by force, a ruler who threatens the safety of true religion. Also in 1554 Knox published his Faithful Admonition to the Protestants who remained in England. Its extremism and intemperate language served to increase the sufferings of those to whom it was addressed; and, coming as it did from one who was in comparative safety, it alienated many in England from him.

In the same year, on the insistence of John Calvin (Calvin, John), Knox became minister of a congregation of English refugees, mainly Puritan, in Frankfurt am Main; but he remained there for only a few months. He then became minister of the growing congregation of English exiles in Geneva, a pastorate that lasted until his final return to Scotland in 1559, but was interrupted at the outset by a visit (1555–56) to Berwick and a nine-month sojourn in Scotland, in the course of which he married Marjorie Bowes. She died, having borne him two sons, in 1560.

In Edinburgh Knox was astounded by the progress made by the Reformed cause and by the eager reception given to him by all classes in the community. To the nobility, in visits to their country houses, he propounded his doctrine of “justifiable resistance” to Roman Catholic rulers who attacked the faith of Protestant subjects and urged them to withdraw from all the rites and ceremonies of the Roman Church and to band themselves together for the defense of Protestantism in case that should prove necessary. A peremptory summons from his congregation called him back to Geneva; but he left to the faithful in Scotland an important Letter of Wholesome Counsel (1556) enjoining not only private family worship but also weekly meetings of believers for corporate Bible study and discussion. From these weekly meetings, Reformed congregations grew apace, and from the leaders of these congregations came the elders of the Reformed Church.

Geneva was to Knox a beloved city in which he spent the happiest years of his life, highly esteemed, in peace, and among kindred souls. From this period (1556–58) dates his elaborate and rather tedious treatise on Predestination as well as his of women, in which he states with uncurbed vehemence the common belief of his day that the exercise of authority by women is contrary to both natural law and revealed religion. The pamphlet was aimed at the three women who were holding the reins of government in England, France, and Scotland and were oppressing Protestantism; but, unfortunately for Knox, publication coincided with the accession in England of the Protestant Elizabeth I, who indignantly and permanently debarred the rash author from her realm.

In Scotland, matters reached a crisis in the spring of 1559. Two years earlier the Protestant lords had signed a “band,” or covenant, on Knox's advice, pledging themselves to foster and defend “the Congregation of the Lord” and its ministers (hence their name “Lords of the Congregation”). The queen regent, the French-born Mary of Guise, had deemed it politic to make concessions to them. But when hostilities between Spain and France ended early in 1559, opening the possibility of stronger French intervention in Scotland, the queen regent felt that the time had come to call a final halt to the expansion of Protestantism. To this end she summoned the Protestant preachers, as ringleaders of the growing Protestant insubordination, to appear before her on May 10 at Stirling. The Protestants replied by recalling Knox from Geneva, and the Protestant lords, lairds, and commoners mustered at Dundee. On May 4, Knox joined them and they advanced to Perth, where, after a vehement sermon by Knox, the friaries were sacked.

By the end of June, Edinburgh was temporarily in Protestant hands and Knox was preaching in St. Giles's; but the triumph was illusory and Knox knew it. The voluntary army of Protestants could not keep the field for more than a few weeks; the mercenary army of the queen regent could keep the field indefinitely and strike a crushing blow as Protestant strength declined. At this juncture Henry II of France died and power fell into the hands of the Guises, the brothers of the queen regent and uncles of the young queen of France— Mary, Queen of Scots and consort of Francis II, the new king of France. Strong French intervention in Scotland was now assured in furtherance of the Guise plan to displace Queen Elizabeth of England and to unite France, Scotland, and England under Francis II, of France, and Mary. Thus a political issue of critical international importance cut athwart the religious issue in Scotland. A French victory in Scotland would place Elizabeth and England in peril. It therefore behooved England to make common cause with the Scottish Protestants. Knox lost no opportunity to drive this fact home to Elizabeth. The autumn and winter of 1559 saw the Scottish Protestants in desperate plight. Only Knox's superhuman exertions and indomitable spirit kept the cause in being. In the blackest hour Knox put fresh heart into the despairing Protestant leaders and staved off defeat at the hands of the government's French mercenaries. On Knox's resolution alone in these months hung the fate not only of Scottish Protestantism but of Elizabeth's England as well.

In the spring of 1560, Elizabeth at last consented to English action. In April, 10,000 English troops joined the Scottish Protestants, the queen regent died in Edinburgh castle, and the disheartened French gave up. By treaty, French and English troops were then withdrawn, leaving the victorious Scottish Protestants to set their own house in order. Queen Mary was a Roman Catholic and an absentee in France, and all her sympathies were with the defeated side. The Scottish Parliament had never exercised much power, but now, meeting in August without royal authority, it proceeded to grapple with the religious issue. The Scots Confession (hurriedly prepared by Knox and three others) was adopted, and papal jurisdiction was abolished.

Knox, aided by a committee of distinguished churchmen, laid before the Scottish Parliament the First Book of Discipline containing proposals for the constitution and finance of the Reformed church. Worship was to be regulated by the Book of Common Order (also called Knox's Liturgy), according to which congregations were to be governed by elders elected annually by the people and the elders were to aid the minister to maintain firm moral discipline among the people. Ministers were to be elected by the people but to be appointed only after rigorous examination of life and doctrine by their ministerial brethren. The ablest ministers were to be appointed superintendents of areas roughly corresponding to the old dioceses; they were to supervise the ministers and congregations in the area and were to be assisted by provincial synods of ministers and elders. In the high place given to the laity, Knox's system contains the most essential element of later Presbyterianism.

The Book of Discipline proceeds to outline a most elaborate educational scheme and plans for a much-needed scheme of systematic aid of the poor. Finally it urges that the endowments of the old church should be made available for the financing of these admittedly costly schemes of the new church. But the proposals thus outlined foundered on the rock of finance. The endowments of the old church were plunder in a poor land for the nobility, who had scant sympathy with Knox's “devout imaginings.” Parliament shelved the financial problem by the temporary expedient of granting to the remaining Roman Catholic clergy the life-rent of their benefices, provided they contributed to the maintenance of the Reformed Church out of their revenues. Knox was deeply embittered by the enforced abandonment of his schemes for education and poor relief and by the scant provision for the Reformed Church.

Mary arrived in Scotland in 1561 already persuaded that Knox was to be her archenemy and that the country could not hold them both. Knox, who hoped at first that the young queen would prove pliable, soon reached a similar conviction. The first three of his audiences with Mary were polite skirmishes; in the fourth, battle was joined in grim earnest. Hearing that Mary was contemplating marriage with Don Carlos of Spain, a match that would have had fatal consequences for the Scottish Reformation and probably for England as well, Knox sounded the Protestant alarm. Mary, enraged at this intervention by a heretic preacher and commoner in affairs of state, berated Knox with hysterical fury and charged him with treason, but the Privy Council refused to convict him. Knox filled Mary's cup of bitterness in 1564 by marrying, without the royal assent, Margaret Stewart, a 17-year-old distant relative of the queen.

In 1564 Mary dismissed her Protestant advisers and undertook the mismanagement of her own affairs. For a time the Reformed Church was in real danger, but in 1567 came Mary's ruin and abdication, and Knox's old friend James Stewart, earl of Moray (Moray, James Stewart, 1st Earl of, Earl Of Mar, Lord Abernethy), became regent. In him the Reformed Church would have found a powerful patron, but he was murdered and the country plunged into a struggle between the supporters of the queen and those of the regency. Knox was involved in the turmoil, but he suffered a paralytic stroke. When Edinburgh became a battleground between the factions in 1571, the leaders on both sides insisted on his removal to safety in St. Andrews, from where he returned in 1572 to die. When the news of the St. Bartholomew's Day Massacre of French Protestants reached Scotland, Knox dragged himself to his pulpit in St. Giles's and drove home the lesson of that tragedy. He stood one last time in the pulpit of St. Giles's, to introduce his successor.

Knox was a controversial figure, and his influence will always be variously assessed by men of differing religious and political views. Certainly his conviction that the Reformation was God's cause and must triumph, a conviction he had a remarkable power of impressing upon other minds, was the rock upon which the Reformed Church in Scotland was built. His power as a preacher lay in his capacity to fuse reason with emotion and to be a passionate logician in the pulpit. Intolerant he undoubtedly was, but his Calvinism was a good deal more moderate than that of a later age. He was more temperate in action than in speech, and his private letters reveal an unexpected tenderness. His single-minded, lifelong, and incorruptible devotion to what he believed to be his duty must command respect. There is ample historical testimony that his moral life was in keeping with his rigorous creed.