Further information: UB Iwerks was considered by many to be Walt Disney's oldest friend, and spent most of his career with Disney. The two met in 1919 while working for the Pesmen-Rubin Art Studio in Kansas City, and would eventually start their own commercial art business together, Disney decided to take up work in animation, and Iwerks soon joined him.

He was responsible for the distinctive style of the earliest Disney animated cartoons, and was also responsible for designing Mickey Mouse.

The first few Mickey Mouse and Silly Symphonies cartoons were animated almost entirely by Iwerks, including Steamboat Willie and The Skeleton Dance. However, as Iwerks began to draw more and more cartoons on a daily basis, he soon found himself unable to cope under Disney's harsh command; Iwerks also felt he wasn't getting the credit he deserved for drawing all of Walt's successful cartoons. Eventually, Iwerks and Disney had a falling out; their friendship and working partnership were severed when Iwerks accepted a contract with Disney competitor Pat Powers to leave Disney and start an animation studio under his own name. The Iwerks Studio opened in 1930.

From 1933 to 1936, he produced a series of shorts in Cinecolor, named ComiColor Cartoons. The ComiColor series mostly focused on fairy tales with no continuing character or star. Later in the 1940s, this series would receive home-movie distribution by Castle Films. Cinecolor produced the 16mm prints for Castle Films with Red emulsion on one side and Blue emulsion on the other. Later in the 1970s Blackhawk Films released these for home use, but this time using conventional Eastmancolor film stock. After his return to the Disney studio, Iwerks mainly worked on developing special visual effects. He is credited as developing the processes for combining live action and animation used in Song of the South (1946), as well as the xerographic process adapted for cel animation. He also worked at WED Enterprises, now Walt Disney Imagineering, helping to develop many Disney theme park attractions during the 1960s. Iwerks did special effects work outside the studio as well, including his Academy Award nominated achievement for Alfred Hitchcock's The Birds (1963).