Robert became head of the firm in 1526, and it was he who adopted the device of the olive tree for his title pages. In 1527–28 he published his first complete Bible in Latin, and in 1531 he completed his great Dictionarium seu linguae latinae thesaurus, a Latin dictionary that marks an epoch in the history of lexicography, not only for Latin but also for all other languages. Francis I of France made him king's printer for Hebrew and Latin works in 1539; in 1540 he became de facto king's printer also for Greek. He was commissioned in 1541 to supply the king's library with books printed in the Greek type of Claude Garamond. He prepared the first printed editions of many works by ancient Greek and Roman authors. Among his Latin editions, his Virgil of 1532 is noteworthy. He also published grammars and other educational texts.

The hostility of theologians at the Sorbonne forced Estienne to leave Paris for Geneva in 1551. His younger brother Charles (d. c. 1564), better known as a writer, succeeded to control of the family press at Paris. Works printed by Robert Estienne in Geneva include a Greek New Testament (1551), in which the present division of the text into verses occurs for the first time.