A distinguished canon lawyer, he was papal envoy to Spain for Pope Clement VIII, who made him cardinal in 1596. He became vicar of Rome in 1603 and on May 16, 1605, was elected as Pope Leo XI's successor at a time when the Kingdom of Naples and the Venetian Republic were violating ecclesiastical rights.

One of his first acts was to excommunicate the recalcitrant minister of Naples for violating the privilegium fori—i.e., the right of ecclesiastics to be judged in criminal cases not by civil courts but by church courts. In 1606 a conflict erupted between Paul and Venice over papal jurisdiction and ecclesiastical immunity within the republic, where the celebrated theologian Paolo Sarpi (Sarpi, Paolo) encouraged resistance to papal censures. The situation became critical when Paul's interdict against Venice (May 1606) caused firmer defiance, led chiefly by Sarpi. Fear of Venice's breaking with Rome and the risk of civil war in Italy induced the neighbouring states to intervene. Paul was prepared to appeal to arms, but a compromise was reached on April 21, 1607, mainly through France's mediation. Paul lifted the interdict and excommunicated Sarpi, against whom a murderous attack was made in the following October. Sarpi accused the Curia of instigating the assault, which Paul reprobated. He realized that the effect of interdicts was dead, and they were not used by the papacy against a sovereign state again.

Earlier (Sept. 22, 1606), Paul had expressly forbidden the Roman Catholics of England to take the new oath of allegiance imposed on them by King James I. His contention with Venice, however, made him politically cautious, and he endeavoured to maintain peace between the Habsburgs and France. He considered another crusade against the Turks, though without success. He particularly feared an open breach of the Peace of Augsburg, the first permanent legal basis for the coexistence of Lutheranism and Catholicism in Germany. Thus, when in 1618 hostility between German Catholics and Protestants caused fighting that developed into the Thirty Years' War, Paul gave no support to the Catholic powers.

Although he censured Galileo and placed Copernicus's treatise on the heliocentric theory of the solar system on the Index of Forbidden Books (Index Librorum Prohibitorum), in doctrinal matters he was surprisingly undogmatic. He encouraged missions, notably those in Latin America, and confirmed many new congregations and brotherhoods, including St. Philip Neri's Oratorians (approved 1613), a congregation of secular priests. He also approved the use of the vernacular in the liturgy for China.To preserve papal documents he founded the privy Vatican archives. In 1612 he authorized a new version of the Rituale Romanum, one of the Roman rite's liturgical books, which he promulgated on June 17, 1614.

Paul was guilty, however, of nepotism and is responsible for his family's inordinate wealth. He especially favoured his nephew Marcantonio Borghese, whom he created prince of Vivaro. His excessive fondness for display, which wasted funds needed for more crucial purposes, made him a spectacular patron of the arts and of building, including the chapel in the Basilica of Santa Maria Maggiore, Rome, where he is buried.