Member of a prominent family and brother of Guillaume du Bellay, Jean du Bellay was made bishop of Bayonne in 1526, a privy counsellor in 1530, and bishop of Paris in 1532. Francis I also employed him as a diplomat, sending him on five missions to England between September 1527 and January 1534 and to Rome in 1534 to defend the English king Henry VIII's divorce. He was made a cardinal in May 1535. In February 1536 he was appointed lieutenant general of the kingdom during the war of 1536–37 between Francis I and the Holy Roman emperor Charles V. During the last years of Francis' reign, du Bellay received many benefices.

On the accession of King Henry II (1547), du Bellay fell into disfavour. He retired to Rome (1553), where he became bishop of Ostia and dean of the Sacred College of Cardinals (1555).

Du Bellay helped his friend Guillaume Budé persuade Francis I to found the Collège de France. The writer François Rabelais was his secretary and doctor; and other men of letters, such as Étienne Dolet and the poet Salmon Macrin, were indebted to him for assistance. His own writings include three books of Latin poems and a defense of Francis I (1542). His correspondence is preserved in the Bibliothèque Nationale in Paris.