Weber was born in Allegheny, Pennsylvania (now Pittsburgh's Northside neighborhood), where she was apparently an excellent pianist. She ran away from home hoping to pursue a singing career in New York City. After leaving home she lived in poverty and worked as a street-corner evangelist, preaching and singing hymns in New York and Pittsburgh. In 1905 she joined the Gaumont Film Company as an actor, and in 1906 married Gaumont manager Phillips Smalley.
In 1908 she landed a role in a film she had written called Hypocrites, which was directed by Herbert Blaché, husband of famous early filmmaker Alice Guy. Hypocrites was also the title of a 1915 film that Weber wrote, directed, produced -- and starred in -- which addressed social themes and moral lessons considered daring for the time. These films (and themes) included abortion and birth control in Where Are My Children?, capital punishment in The People vs. John Doe, and alcoholism and drug addiction in Hop, the Devil's Brew (all 1916). Because of their controversial nature, her films were often successful at the box office.
In 1916 she became Universal Studios' highest-paid director, and in 1917 she formed her own production company, Lois Weber Productions. Lois Weber was the only woman granted membership in the Motion Picture Directors Association. Film director John Ford worked with Weber as her assistant before making films on his own. One of Weber's most successful films from this period was The Blot (1921) with Claire Windsor and Louis Calhern, one of five films of Weber's released through Paramount Pictures.
In the 1920s her fortunes began to change - she lost her company, obtained a divorce from the abusive, alcoholic Smalley in 1922, and had a nervous breakdown. She married Harry Gantz in 1926, but they divorced in 1935. Her last silent films were Sensation Seekers and The Angel of Broadway (both 1927). Weber's final film, and her only talkie, was Heat (1934), a tale of miscegenation on a sugar plantation. The film was poorly received. She could later could only find work as a script doctor for Universal Studios.
She died, penniless, at the age of 58. She had no children.
For her contribution to the motion picture industry, Lois Weber has a star on the Hollywood Walk of Fame at 6518 Hollywood Blvd.