In 1942 O'Connor joined Universal Pictures' troupe of talented teenagers. He received gradually larger roles in four of the studio's Gloria Jean musicals, and achieved stardom at 17 with Mister Big (1943), co-starring Gloria Jean and comic dancer Peggy Ryan. O'Connor and Ryan's energetic routines invited comparisons with M-G-M's pairing of Mickey Rooney and Judy Garland.
O'Connor was drafted into the armed forces in 1944. Before he reported for duty, Universal rushed him through the production of three feature films, produced simultaneously and released while the actor was overseas. Upon his return, Universal (now reorganized as Universal-International) cast him in lightweight musicals and comedies. In 1949, he was given the leading role in Francis, the whimsical story of a sad-sack soldier befriended by a talking mule. The film was a huge success, and a mixed blessing for O'Connor: the momentum of his musical career was constantly interrupted because the studio insisted on his making one "Francis" picture a year until 1955. It was because of Francis that O'Connor missed out on a plum role: Bing Crosby's sidekick in White Christmas. O'Connor was forced to bow out when he contracted an illness transmitted by the mule. He was replaced in the film by Danny Kaye. But, still he would manage to make film history with Singin' in the Rain. His role as Cosmo the piano player would earn him the Golden Globe award for Best Performance by an Actor in a Comedy or Musical.
Donald O'Connor was a TV favorite in the 1950s, and was one of the regular hosts of NBC's popular Colgate Comedy Hour. He hosted a color television special on NBC in 1957, which was among the first color programs to be videotaped; an excerpt of the telecast was included in NBC's 50th anniversary special in 1976. He also had a short-lived television series during the late 1960s.
After overcoming a drinking problem in the 1970s, he had a huge career boost when he hosted the Oscars, which earned him two Primetime Emmy nominations. He appeared as a gaslight-era entertainer in the 1981 film Ragtime, notable for similar encore performances by James Cagney and Pat O'Brien. O'Connor also appeared in the short-lived Bring Back Birdie on Broadway in 1981, and continued to make film and television appearances into the 1990s. Donald O'Connor's last feature film was the Jack Lemmon-Walter Matthau comedy Out to Sea, in which he played a dance host on a cruise ship. O’Connor was still making public appearances well into 2003.
He left behind his wife of over 40 years, Gloria, and four children.