After a false start headlining an obscure 1938 musical film called, The Duke is Tops, Horne became the first African American performer to sign a long-term contract with a major Hollywood studio, namely Metro-Goldwyn-Mayer. She made her debut with MGM in 1942's Panama Hattie and became famous in 1943 for her rendition of Stormy Weather in the movie of the same name (which she made while on loan to 20th Century Fox from MGM). She appeared in a number of MGM musicals, most notably Cabin in the Sky (also 1943), but was never featured in a leading role due to her race and the fact that films featuring her had to be reedited for showing in southern states where theatres could not show films with African American performers. As a result, most of Horne's film appearances were standalone sequences that had no bearing on the rest of the film, so editing caused no disruption to the storyline; a notable exception was the all-black musical Cabin in the Sky, though even then one of her numbers had to be cut because it was considered too suggestive by the censors. She was originally considered for the role of Julie LaVerne in the 1951 version of Show Boat but Ava Gardner was given the role instead (the production code office had banned interracial relationships in films).

Horne was married to Lennie Hayton, a white, Jewish American for many years until his death. Hayton was one of the premier musical conductors and arrangers at MGM. Studio executives disapproved of the marriage, and eventually both Horne and Hayton were let go. In her as-told-to autobiography Lena by Richard Schickel, Horne recounts the enormous pressures she and her husband faced as an interracial married couple.

Disenchanted with Hollywood by the mid-1950s, and increasingly focused on her nightclub career, she only made two major appearances in MGM films during the decade, 1950's Duchess of Idaho (which was also Eleanor Powell's film swan song), and the 1956 musical Meet Me in Las Vegas. She returned to the screen three more times, playing Claire Quintana in the 1969 film Death of a Gunfighter, Glinda the Good Witch in The Wiz (1978), with Diana Ross and Michael Jackson, and co-hosting the 1994 MGM retrospective That's Entertainment! III.

She appeared in Broadway musicals several times and in 1958 was nominated for the Tony Award for "Best Actress in a Musical." In 1981 she received a Special Tony Award for her show, Lena Horne: "The Lady and Her Music". She also made occasional TV appearances, such as a guest appearance as herself on Sanford and Son in the 70s and a mid-1980s performance on The Cosby Show.

In 2003, ABC announced that pop star Janet Jackson would star as Horne in a television biopic. In the weeks following Jackson's so-called "wardrobe malfunction" debacle during the 2004 Super Bowl, however, Variety reported that Horne demanded Jackson be dropped from the project. "ABC executives resisted Horne's demand," according to the Associated Press report, "but Jackson representatives told the trade newspaper that she left willingly after Horne and her daughter, Gail Lumet Buckley, asked that she not take part." Oprah Winfrey told Alicia Keys during a 2005 interview on The Oprah Winfrey Show that she might possibly consider producing the biopic herself, and casting Keys as Horne.

In January 2005, Blue Note Records, her label for more than a decade, announced that "the finishing touches have been put on a collection of rare and unreleased recordings by the legendary Horne made during her time on Blue Note. Remixed by her longtime producer Rodney Jones, the recordings sound wonderful and include versions of such signature songs as 'Something To Live For', 'Chelsea Bridge' and 'Stormy Weather'." The album, originally titled Soul but renamed Seasons of a Life, was recorded in 1999 but remained unreleased for six years. The album was released on January 24, 2006.