He was born at Troyes. From childhood he loved literature, and his father Pierre encouraged this interest. Young Pithou was called to the Paris bar in 1560. On the outbreak of the second war of religion in 1567, Pithou, who was a Calvinist, withdrew to Sedan, France and afterwards to Basel, returning to France on the publication of the edict of pacification. Soon afterwards he accompanied the duc de Montmorency on his embassy to England, returning shortly before the massacre of St Bartholomew, in which he narrowly escaped with his life. Next year he followed the example of the future Henry IV of France by abjuring the Protestant faith.
Henry, shortly after his accession to the throne, recognized Pithou's talents and services by giving him various legal appointments. The most important work of his life was his co-operation in the production of the Satire Ménippée (1593), which did so much to damage the cause of the Catholic League; the harangue of the Sieur d'Aubray is usually attributed to Pithou. He died at Nogent-sur-Seine. His valuable library, specially rich in manuscripts, was for the most part transferred to what is now the Bibliothèque Nationale in Paris.
Pithou wrote many legal and historical books, besides preparing editions of several ancient writers. His earliest publication was Adversariorum subsectorum lib. II. (1565). In 1569, he became the first to publish Landolfus Sagax' Historia Romana, and under the name by which it became better known: Historia Miscella. Perhaps his edition of the Leges Visigothorum (1579) was his most valuable contribution to historical science; in the same line he edited the Capitula of Charlemagne, Louis the Pious, and Charles the Bald in 1588, and he also assisted his brother François in preparing an edition of the Corpus juris canonici (1687). His Libertés de l'église gallicane (1594) is reprinted in his Opera sacra juridica his orica miscellanea collecta (1609). In classical literature he was the first who made the world acquainted with the Fables of Phaedrus (1596); he also edited the Pervigilium Veneris (1587), and Satires of Juvenal and Persius (1585).