Burt Reynolds as Charlie B. Barkin, a brash German Shepherd and a former con artist. The character was designed specifically with Reynolds in mind for the role and the animators mimicked some of his mannerisms.
Reynolds is succeeded by Charlie Sheen for All Dogs Go to Heaven 2, and Steven Weber for All Dogs Go to Heaven: The Series and An All Dogs Christmas Carol. Charlie was modeled after a German Shepherd, appropriately named Burt. Burt the dog often spent time with the animators at the studio, even going with them during the studio's move to Ireland.
Dom DeLuise as Itchy Itchiford, a paranoid, anxious but loyal Dachshund and Charlie's best friend. This was the sixth film in which DeLuise co-starred with Reynolds.
DeLuise reprised his role in All Dogs Go to Heaven 2, the series and An All Dogs Christmas Carol.
Judith Barsi as Anne-Marie, a 7-year-old orphan girl with the ability to talk to and understand animals. Her singing voice was performed by Lana Beeson. This was Barsi's final film role before her murder in 1988. The end credits song "Love Survives" was dedicated in her memory and her character did not appear in any subsequent media.
Vic Tayback as Carface Carruthers, a violent, sadistic Bulldog gangster. This was Tayback's final film role before his death in 1990.
For All Dogs Go to Heaven 2, All Dogs Go to Heaven: The Series and An All Dogs Christmas Carol, Ernest Borgnine took Tayback's role.
Charles Nelson Reilly as Killer, a misnamed, fidgety, neurotic and spectacles-wearing Schnoodle who is Carface's comic relief sidekick.
Reilly was the second original voice actor that reprised his role in the series and An All Dogs Christmas Carol except for the sequel.
Loni Anderson as Flo, a female Rough Collie and one of Charlie's friends.
Melba Moore as a Whippet angel who welcomes deceased dogs into Heaven. She was named "Annabelle" in the 1996 sequel and its subsequent syndicated television series.
Bebe Neuwirth succeeded Moore in this role.
Ken Page as King Gator, an American alligator and voodoo witch doctor living below the streets of New Orleans.
Rob Fuller and Earleen Carey as Kate and Harold, a kindly married couple who later become Anne-Marie's adoptive parents.
Godfrey Quigley as Terrier, a dog that appears when Itchy tells everyone Anne-Marie is in danger.
Anna Manahan as Stella Dallas, a horse that appears when Anne-Marie, Charlie and Itchy are at the derby. She is against the Once Upon a Wintertime Team.
Candy Devine as Vera, a female gambling dog.
Dan Molina (uncredited) as the hellhound in Charlie's nightmare and at the finale. It is depicted in the film as a canine analog of Devil.