Culhane worked for a number of American animation studios, including Fleischer Studios, the Ub Iwerks studio, Walt Disney Productions, and the Walter Lantz studio. He began his animation career in 1925 working for J.R. Bray studios.
While at the Disney studio, he was a lead animator on Snow White and the Seven Dwarfs, animating arguably the most well-known sequence in the film: the animation of the dwarves marching home singing "Heigh-Ho". The scene took Culhane and his assistants six months to complete. During this period he worked under the nickname James Culhane.
Later in his career, Culhane was a director for Lantz, where he helmed Woody Woodpecker's 1944 classic, The Barber of Seville. In the late-1940s, he founded Shamus Culhane Productions, one of the first companies to create animated television commercials.
Shamus Culhane Productions folded in the 1960s, at which point Culhane became the head of the successor to Fleischer Studios, Paramount Cartoon Studios. He left the studio in 1967, and went into semi-retirement.
Culhane wrote two highly-regarded books on animation: the how-to/textbook Animation from Script to Screen, and his autobiography Talking Animals and Other People. Since Culhane worked for a number of major Hollywood animation studios, his autobiography gives a balanced general overview of the history of the Golden Age of American Animation.
Married twice, Culhane's first wife was Maxine Marx, Chico Marx's daughter, with whom he had two sons. At his death on February 2, 1996, Culhane was also survived by his second wife, Juana Hegarty, to whom he was married for 35 years.