He was born at Montbard on the Côte-d'Or. He studied under Cavelier, Millet, and Barrias, at the École des Beaux-Arts, which he entered in 1841, and where he gained the prix de Rome in 1845 with Theseus finding on a rock his father's sword. He became director of the École des Beaux-Arts in 1864, and director-general of Fine Arts from 1878–79, when the office was suppressed.
Guillaume was a prolific writer, principally on sculpture and architecture of the Classic period and of the Italian Renaissance. He was elected member of the Académie française in 1898, and in 1891 was sent to Rome as director of the Académie de France in that city. He held this position until 1904. He was also elected an honorary member of the Royal Academy, London, 1869, on the institution of that class.