- Anthony Perkins as Norman Bates
- Vera Miles as Lila Crane
- John Gavin as Sam Loomis
- Janet Leigh as Marion Crane
- Martin Balsam as Detective Milton Arbogast
- John McIntire as Sheriff Al Chambers
- Simon Oakland as Dr. Fred Richman
- Frank Albertson as Tom Cassidy
- Pat Hitchcock as Caroline
- Vaughn Taylor as George Lowery
- Lurene Tuttle as Mrs. Chambers
- John Anderson as California Charlie
- Mort Mills as Highway Patrol Officer
- Francis De Sales as Deputy District Attorney Alan Deats (uncredited)
- George Eldredge as Police Chief James Mitchell (uncredited)
- Ted Knight as a policeman guarding Norman Bates (uncredited)
- Virginia Gregg, Paul Jasmin, and Jeanette Nolan as the voice of Norma Bates (uncredited)
The success of Psycho jump-started Perkins' career, but he soon began to suffer from typecasting. However, when Perkins was asked whether he would have still taken the role knowing that he would be typecast afterwards, he replied with a definite "yes".
Until her death, Leigh continued to receive strange and sometimes threatening calls, letters, and even tapes detailing what they would like to do to Marion Crane. One letter was so "grotesque" that she passed it along to the FBI, two of whose agents visited Leigh and told her the culprits had been located and that she should notify the FBI if she received any more letters of that type.
Norman's mother was voiced by Virginia Gregg, Paul Jasmin, and Jeanette Nolan, who also provided some screams for Lila's discovery of the mother's corpse. The three voices were thoroughly mixed, except for the last speech, which is all Gregg's.
As Perkins was in New York working on a Broadway stage show when the shower sequence was filmed, actresses Anne Dore and Margo Epper stepped in as his body doubles for that scene.