- Anthony Perkins as Norman Bates
- Janet Leigh as Marion Crane
- Vera Miles as Lila Crane
- John Gavin as Sam Loomis
- Martin Balsam as Detective Milton Arbogast
- John McIntire as Al Chambers
- Simon Oakland as Dr. Fred Richmond
- Frank Albertson as Tom Cassidy
- Pat Hitchcock as Caroline
- Vaughn Taylor as George Lowery
- Lurene Tuttle as Mrs. Chambers
- John Anderson as California Charlie (used car salesman)
- Mort Mills as Highway Patrol Officer
- Virginia Gregg, Jeanette Nolan, and Paul Jasmin as voice of Norma Bates
- Ted Knight as a policeman guarding Norman 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.