Katharine Hepburn as Mary Stuart

Florence Eldridge as Elizabeth Tudor

Fredric March as Bothwell

Douglas Walton as Darnley

John Carradine as Rizzio

Robert Barrat as Morton

Gavin Muir as Leicester

Ian Keith as Moray

Moroni Olsen as John Knox

William Stack as Ruthven

Ralph Forbes as Randolph

Alan Mowbray as Throckmorton

Frieda Inescort as Mary Beaton

Donald Crisp as Huntly

David Torrence as Lindsay

Molly Lamont as Mary Livingstone

Anita Colby as Mary Fleming

Jean Fenwick as Mary Seton

Lionel Pape as Burghley

Alec Craig as Donal

Mary Gordon as Nurse

Monte Blue as Messenger

Robert Warwick as Sir Francis Knollys

Halliwell Hobbes as Man (uncredited)

Nigel De Brulier as Judge