Bela Lugosi as Scientist

Edward D. Wood Jr. as Glen / Glenda (as Daniel Davis)

Lyle Talbot as Inspector Warren

Timothy Farrell as Dr. Alton / Narrator

Dolores Fuller as Barbara

'Tommy' Haynes as Alan / Anne

Charlie Crafts as Johnny (as Charles Crafts)

Conrad Brooks as Banker / Reporter / Pickup Artist / Bearded Drag (as Connie Brooks)

Henry Bederski as Man with Hat and Receding Hairline (uncredited)

Carol Daugherty as Woman in Nightmare (uncredited)

'Captain' DeZita as The Devil / Glen's Father (uncredited)

Helen Miles as (uncredited)

Shirley Speril as Miss Stevens (uncredited)

Amzie Strickland as Minor Role (uncredited)

Harry Thomas as Man in Nightmare (uncredited)

William C. Thompson as Judge (uncredited)

'Mr. Walter' Hadjwiecyz as Patrick / Patricia (uncredited)

George Weiss as Man at Transvestite's Suicide (uncredited)

Evelyn Wood as Sheila, Glen's Sister (uncredited)