Wayne Allwine, 62, died on Monday of complications from diabetes, the Walt Disney Company said.
"Wayne dedicated his entire professional life to Disney," the Disney chief executive Robert Iger said. "Over the last 32 years, he gave so much joy, happiness and comfort to so many around the world by giving voice to our most beloved, iconic character, Mickey Mouse."
Born in Glendale, California, Allwine joined Disney in 1966 when he took a job in the mail room. He went on to work in the sound effects department and began voicing the company’s main mouse in 1977 after using his natural falsetto to win an open audition.
He was only the third man behind Mickey's voice. The first was Walt Disney himself, who spoke for the mouse from 1928 to 1947, before passing the role to Jimmy MacDonald, the studio's sound effects wizard. MacDonald had already been Allwine's mentor in the studio and told him after passing on the reins: "Just remember kid, you're only filling in for the boss."
"Mickey's the real star," Allwine said later. "You know you just have to love the little guy while you have him, because he won’t be yours forever."
Allwine's first voice work as Mickey was for television but also provided the mouse's voice for movies including "Mickey's Christmas Carol" (1983), "Who Framed Roger Rabbit", (1988), and "The Prince and the Pauper" (1990) and for Disney theme parks, radio and live stage events.
Allwine is survived by his wife, Russi Taylor, whom he married in Hawaii in 1981, and is one of Hollywood's most famous voice actresses. She has been the voice of Minnie since 1986.