Katharine Hepburn
Mary Stuart

Florence Eldridge
Elizabeth Tudor

Fredric March
Bothwell

Douglas Walton
Darnley

John Carradine
Rizzio

Robert Barrat
Morton

Gavin Muir
Leicester

Ian Keith
Moray

Moroni Olsen
John Knox

William Stack
Ruthven

Ralph Forbes
Randolph

Alan Mowbray
Throckmorton

Frieda Inescort
Mary Beaton

Donald Crisp
Huntly

David Torrence
Lindsay

Molly Lamont
Mary Livingstone

Anita Colby
Mary Fleming

Jean Fenwick
Mary Seton

Alec Craig
Donal

Mary Gordon
Nurse

Monte Blue
Messenger

Robert Warwick
Sir Francis Knollys

Halliwell Hobbes
Man (uncredited)

Nigel De Brulier
Judge