Local delivery of mrIFN-β provides innate protection against vaginal HSV-2 challenge of B6 and IRF-3−/− mice. Six- to eight-week-old female IRF-3−/− mice (n = 8) or control B6 mice (n = 10) were treated with cell supernatants containing mrIFN-β (mrIFN-β sup) or mock supernatants (mock sup) or left untreated. Twenty-four hours posttreatment, mice were challenged with IVAG HSV-2. Challenged mice were monitored daily for genital pathology and survival. Local delivery of mrIFN-β provided B6 mice with 80% protection and IRF-3−/− mice with 75% protection against subsequent IVAG HSV-2 challenge. Mice treated with mock supernatants showed no protection against IVAG HSV-2 challenge compared to naïve mice.