Jowett was educated at St. Paul's School, London, and Balliol. He was made a fellow at Balliol in 1838 and was appointed a tutor in the college in 1842, the year in which he was ordained an Anglican deacon. He was ordained priest three years later.

In 1855 Jowett finished his book The Epistles of St. Paul. His essay on the atonement was attacked as unorthodox, but in spite of strong opposition he was appointed regius professor of Greek, with an annual salary of £40. His contribution to Essays and Reviews (1860) caused his opponents to accuse him of heresy before the vice chancellor's court, but proceedings were eventually dropped. Nevertheless, attempts to augment his salary were opposed, and it was not until 1865 that Christ Church freed endowments to produce a stipend of £500 a year. During this period his lectures on the Republic stimulated intense interest in Plato.

Jowett's election as master of Balliol in 1870 enabled him to rebuild a considerable portion of the college and to establish a hall for noncollegiate students. During this period he published his translations of The Dialogues of Plato (1871) and of Thucydides' History (1881). As vice-chancellor of the university (1882–86), he encouraged drama and music and completed his translation of Aristotle's Politics. His edition of the Republic, on which he had worked for 30 years, was published posthumously in 1894.