In 1517, after his wife's death, he took holy orders and gradually rose in the Catholic Church hierarchy: first as bishop of several dioceses held by him in plurality; then as Archbishop of Sens, 1525; cardinal, 1527, and legate a latere, 1530.

French historian Gabriel Hanotaux, in the introduction to his Recueil des instructions, calls Duprat one of the most notable men of ancient France, second only to Richelieu in the decisive influence he exercised on the destinies of his country.This influence was constantly exerted to strengthen royal absolute power; it was felt in the measures he took against the grands Seigneurs and in his elaborate fiscal system.

Duprat's influence was also manifested, together with his orthodoxy, in those measures which affected the relations of France with the Church, namely, the signing of the Concordat of Bologna, and the checking of nascent Protestantism.

The Concordat, which Duprat himself negotiated with Pope Leo X at Bologna, did away with the principles of the "Pragmatic Sanction"; on the other hand, by causing the appointment of the French hierarchy to rest on royal nomination instead of the old canonical elections, it vested in the civil power an authority over church affairs.

The Sorbonne and the Parlement were instructed to exclude the writings of the innovators; in 1534 the posting of subversive pamphlets at the door of the royal apartments cost the perpetrators their lives.