In 1908, Griffith accepted a role as a stage extra in Professional Jealousy for the American Mutoscope and Biograph Company, where he met cameraman Billy Bitzer, and his career in the film industry changed forever. In 1908, Biograph's main Director Wallace McCutcheon, Sr. grew ill, and his son Wallace McCutcheon, Jr. took his place. McCutcheon, Jr. did not bring the studio success; Biograph co-founder Harry Marvin gave Griffith the position, and he made the short The Adventures of Dollie. He directed a total of 48 shorts for the company that year.

Griffith has five films preserved in the United States National Film Registry deemed as being "culturally, historically, or aesthetically significant." These are Lady Helen's Escapade (1909), A Corner in Wheat (1909), The Birth of a Nation (1915), Intolerance: Love's Struggle Throughout the Ages (1916), and Broken Blossoms (1919).

His short In Old California (1910) was the first film shot in Hollywood, California. Four years later, he produced and directed his first feature film Judith of Bethulia (1914), one of the earliest to be produced in the US. Biograph believed that longer features were not viable at that point. According to Lillian Gish, the company thought that "a movie that long would hurt [the audience's] eyes".

Griffith seems to have been the first to understand how certain film techniques could be used to create an expressive language; it gained popular recognition with the release of his The Birth of a Nation (1915). His early shorts—such as Biograph's The Musketeers of Pig Alley (1912), the first "gangster film"—show that Griffith's attention to camera placement and lighting heightened mood and tension. In making Intolerance, the Director opened up new possibilities for the medium, creating a form that seems to owe more to music than to traditional narrative.

Griffith's production partnership was dissolved in 1917 and he went to Artcraft, part of Paramount Pictures, and then to First National Pictures (1919–1920). At the same time, he founded United Artists together with Charlie Chaplin, Mary Pickford, and Douglas Fairbanks. He continued to make films, but he never achieved box office grosses as high as either The Birth of a Nation or Intolerance.

Though United Artists survived as a company, Griffith's association with it was short-lived. While some of his later films did well at the box office, commercial success often eluded him. Griffith features from this period include Broken Blossoms (1919), Way Down East (1920), Orphans of the Storm (1921), Dream Street (1921), One Exciting Night (1922) and America (1924). Of these, the first three were successes at the box office. Griffith was forced to leave United Artists after Isn't Life Wonderful (1924) failed at the box office.

He made a part-talkie, Lady of the Pavements (1929), and only two full-sound films, Abraham Lincoln (1930) and The Struggle (1931). Neither was successful, and after The Struggle he never made another film.

Although mostly forgotten by movie-goers of the time, Griffith was held in awe by many in the film industry. In the mid-1930s, he was given a special Oscar by the Academy of Motion Picture Arts and Sciences. In 1946, he made an impromptu visit to the film location of David O. Selznick's epic western Duel in the Sun, where some of his veteran actors, Lillian Gish, Lionel Barrymore and Harry Carey, were cast members. Gish and Barrymore found their old mentor's presence distracting and became self-conscious. While the two were filming their scenes, Griffith hid behind set scenery.

In 1936, Director Woody Van Dyke, who had worked as Griffith's apprentice on Intolerance, asked Griffith to help him shoot the famous earthquake sequence for San Francisco, but did not give him any film credit. Starring Clark Gable, Jeanette MacDonald and Spencer Tracy, it was the top-grossing film of the year.

Although Griffith eventually disagreed with Roach over the production and parted, Roach later insisted that some of the scenes in the completed film were directed by Griffith. This would make the film the final production in which Griffith was actively involved. But, cast members' accounts recall Griffith directing only the screen tests and costume tests. When Roach advertised the film in late 1939 with Griffith listed as Producer, Griffith asked that his name be removed.

On the morning of July 23, 1948, Griffith was discovered unconscious in the lobby at the Knickerbocker Hotel in Los Angeles, California, where he had been living alone. He died of a cerebral hemorrhage at 3:42 PM on the way to a Hollywood hospital. A large public Service was held in his honor at the Hollywood Masonic Temple, but few stars came to pay their last respects. He is buried at Mount Tabor Methodist Church Graveyard in Centerfield, Kentucky. In 1950, The Directors Guild of America provided a stone and bronze monument for his gravesite.

Griffith left Biograph because of company resistance to his goals and his cost overruns on the film. He took his company of actors with him and joined the Mutual Film Corporation. There, he co-produced The Life of General Villa, a biographical action–drama film starring Pancho Villa as himself, shot on location in México during a civil war. He formed a studio with Majestic Studio manager Harry Aitken which became known as Reliance-Majestic Studios and was later renamed Fine Arts Studio. His new production company became an autonomous production unit partner in Triangle Film Corporation along with Thomas H. Ince and Keystone Studios' Mack Sennett. The Triangle Film Corporation was headed by Aitken, who was released from the Mutual Film Corporation, and his brother Roy.

When Griffith was 14, his mother abandoned the farm and moved the family to Louisville, Kentucky, where she opened a boarding house. It failed shortly after. Griffith then left high school to help support the family, taking a job in a dry goods store and later in a bookstore. He began his creative career as an actor in touring companies. Meanwhile, he was learning how to become a Playwright, but had little success—only one of his plays was accepted for a performance. He traveled to New York City in 1907 in an attempt to sell a script to Edison Studios Producer Edwin Porter; Porter rejected the script but gave him an acting part in Rescued from an Eagle's Nest instead. He then decided to become an actor and appeared in many films as an extra.