Born in Allegheny, Pennsylvania, Gertrude Stein spent her childhood in Vienna and Paris before the family settled in Oakland, California, where her father and her eldest brother Michael made a fortune in street railways and property. After attending Radcliffe College, where she studied with the philosopher William James from 1893 to 1897, she went to Johns Hopkins University in Baltimore to study medicine. In 1902, abandoning a medical career in favour of literature, she followed her brother Leo to Paris and with him established a famous salon. They also began to buy the paintings of Matisse, Braque, and Picasso before these artists were well known, thereby enabling her to take the credit for their later success. Although this was vehemently denied by the artists involved, she nevertheless did much to promote modern art, and Picasso became a long-standing friend.

By 1903 Stein had established a relationship with her lifelong companion, Alice B. Toklas, a friend from San Francisco. Her early writings were directly influenced by the work of such painters as Cézanne, Matisse, and Picasso. Her first important book, Three Lives (1909), was followed by a series of abstract verbal portraits of her friends suggested by early cubist portraits. These led to the composition of her major work, The Making of Americans (1925).

After World War I, during which she and Toklas did fieldwork with the American Fund for French Wounded, Stein was taken up with salon life and the support of expatriate writers. She did not attain wide recognition until 1933, when she published her memoirs, The Autobiography of Alice B. Toklas. In 1934 she returned to the United States for the opening of her opera Four Saints in Three Acts, with a musical score by Virgil Thomson, in Hartford, Connecticut, and gave a highly publicized lecture tour. During World War II she remained in occupied France, an experience she recounted in Wars I Have Seen (1945). In her last years Stein's kindness and generosity made her a great favourite with American servicemen in Paris, and her last novel, Brewsie and Willie (1946), is concerned with them.

Gertrude Stein always meant to return to the United States after the war but died of cancer before she was able to leave Paris. The Mother of Us All, an opera based on the life of Susan B. Anthony, for which Stein provided the libretto and Virgil Thomson the score, was first produced in New York in 1947.