In the fall of 1933, Horne joined the chorus line of the Cotton Club in New York City. In the spring of 1934, she had a featured role in the Cotton Club Parade. A few years later she joined Noble Sissle's Orchestra,
with which she toured. After she separated from her first husband, Horne toured with bandleader Charlie Barnet in 1940–41, but disliked the travel and left the band to work at the Café Society in New York. She replaced Dinah Shore as the featured vocalist on NBC's popular jazz series The Chamber Music Society of Lower Basin Street. The show's resident maestros, Henry Levine and Paul Laval, recorded with Horne in June 1941 for RCA Victor. Horne left the show after only six months to headline a nightclub revue on the west coast; she was replaced by Linda Keene.
Horne already had two low-budget movies to her credit: a 1938 musical feature called The Duke is Tops (later reissued with Horne's name above the title as The Bronze Venus); and a 1941 two-reel short subject, Boogie Woogie Dream, featuring pianists Pete Johnson and Albert Ammons. Horne's songs from Boogie Woogie Dream were later released individually as soundies. Horne was primarily a nightclub performer during this period, and it was during a 1943 club engagement in Hollywood that talent scouts approached Horne to work in pictures. She chose Metro-Goldwyn-Mayer, the most prestigious studio in the world, and became the first black performer to sign a long-term contract with a major Hollywood studio.
She made her debut with MGM in Panama Hattie (1942) and performed the title song of Stormy Weather (1943), which she made at 20th Century Fox, on loan 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 because of her race and the fact that films featuring her had to be re-edited for showing in states where theaters could not show films with black
performers. As a result, most of Horne's film appearances were stand-alone 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, although one number was cut because it was considered too suggestive by the censors. "Ain't it the Truth" was the song (and scene) cut before the release of the film Cabin in the Sky. It featured Horne singing "Ain't it the Truth", while taking a bubble bath (considered too "risqué" by the film's executives). This scene and song are featured in the film That's Entertainment! III (1994) which also featured commentary from Horne on why the scene was deleted prior to the film's release.
In Ziegfeld Follies (1946) she performs "Love" by Hugh Martin and Ralph Blane. Horne wanted to be considered for the role of Julie LaVerne in MGM's 1951 version of Show Boat (having already played the role when a segment of Show Boat was performed in Till the Clouds Roll By) but lost the part to Ava Gardner, a personal friend in real life, due to the Production Code's ban on interracial relationships in films. In the documentary
That's Entertainment! III Horne stated that MGM executives required Gardner to practice her singing using Horne's recordings, which offended both actresses. Ultimately, Gardner's voice was overdubbed by actress Annette Warren (Smith) for the theatrical release, though her voice was heard on the soundtrack album.
Horne was long involved with the Civil Rights movement. In 1941, she sang at Cafe Society and worked with Paul Robeson. During World War II, when entertaining the troops for the USO, she refused to perform "for
segregated audiences or for groups in which German POWs were seated in front of African American
servicemen", according to her Kennedy Center biography. Since the US Army refused to allow integrated audiences,she wound up putting on a show for a mixed audience of black US soldiers and white German POWs. She was at an NAACP rally with Medgar Evers in Jackson, Mississippi, the weekend before Evers was assassinated. She also met President John F. Kennedy at the White House two days before he was assassinated. She was at the March on Washington and spoke and performed on behalf of the NAACP, SNCC
and the National Council of Negro Women. She also worked with Eleanor Roosevelt to pass anti-lynching laws. She was a member of Delta Sigma Theta Sorority, Inc.