Rutherford was born on November 2, 1917, in Vancouver, British Columbia to John Rutherford and Lucille (née Mansfield; 1890–1981) Rutherford. Rutherford's mother was a silent film actress, and her father was a former operatic tenor. While Rutherford was still a baby, the family moved to San Francisco. Soon afterwards, her parents separated and Lucille Mansfield moved to Los Angeles, with Ann and her sister Laurette, who later became known as Judith Arlen.
While roller skating home from middle school in Hollywood, Rutherford would stop at some of the radio studios to listen to voice actors perform. After being criticized one day by her English teacher, Rutherford decided to show her up. The girl falsified an acting history and applied for work at radio station KFAC. A month later, Rutherford had a part in a radio serial drama.
In 1935, Rutherford began her Hollywood film career in the starring role of Joan O'Brien in the dramatic film Waterfront Lady for Mascot Pictures, later to be Republic Pictures. Rutherford soon established herself as a popular leading lady of Western films at Republic, costarring with actors Gene Autry and John Wayne.

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