Gish grew up from roughly 1900 in New York City and made her stage debut at age five. During Lillian and Dorothy's years as child actresses, they formed close friendships with Mary Pickford (Pickford, Mary) (then still known as Gladys Mary Smith), who in 1912 introduced them to Griffith. Immediately struck by their beauty and charm, he gave them small parts in a series of silent movies, beginning with An Unseen Enemy (1912), and the next year placed them under contract to his studio. Almost from the start Lillian was the more popular of the two. An extra measure of winsome appeal in such two-reelers as The Musketeers of Pig Alley (1912), The Mothering Heart (1913), and Judith of Bethulia (1914) won her a large audience of admirers; and after her appearance in The Birth of a Nation (1915), she was established as one of Hollywood's top stars. In Intolerance (1916) and Broken Blossoms (1919) she embodied the ideal of the innocent, vulnerable heroine.

With the coming of the talkies, Lillian left the screen for a time and returned to the stage. With great success, she played on the stage in Uncle Vanya (1930) and subsequently appeared in Camille (1932), Nine Pine Street (1933), Within the Gates (1934), Hamlet (1936), The Old Maid (1936), The Star Wagon (1937), Life with Father (1940, in which she enjoyed a record run in Chicago while Dorothy was starring with the road company), Mr. Sycamore (1942), Magnificent Yankee (1946), Crime and Punishment (1947), The Curious Savage (1950), The Trip to Bountiful (1953), The Family Reunion (1958), All the Way Home (1960), I Never Sang for My Father (1967), and many others. Her last Broadway appearance was in A Musical Jubilee in 1975.

Gish occasionally continued to appear in movies, among them The Commandos Strike at Dawn (1942), Miss Susie Slagle's (1946), Duel in the Sun (1946), The Night of the Hunter (1955), The Unforgiven (1960), The Comedians (1967), A Wedding (1978), Hambone and Hillie (1984), Sweet Liberty (1986), and her final film, The Whales of August (1987), with Bette Davis (Davis, Bette). She also appeared on television in a number of distinguished dramatic presentations, most notably in Arsenic and Old Lace with Helen Hayes (Hayes, Helen) in 1969. Her autobiographical book The Movies, Mr. Griffith, and Me was published in 1969, followed by two more volumes of memoirs, Dorothy and Lillian Gish (1973) and An Actor's Life for Me (1987). She was awarded a special honorary Academy Award in 1971. She also received a lifetime achievement award from the American Film Institute in 1984.