Toll-like receptors (TLRs) constitute a family of innate receptors that recognize and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Previous studies have demonstrated that ligands for TLR3 and TLR9 induce potent innate antiviral responses against herpes simplex virus type 2 (HSV-2). However, the factor(s) involved in this innate protection is not well-defined. Here we report that production of beta interferon (IFN-β) but not production of IFN-α, IFN-γ, or tumor necrosis factor alpha (TNF-α) strongly correlates with innate protection against HSV-2. Local delivery of poly(I:C) and CpG oligodeoxynucleotides induced significant production of IFN-β in the genital tract and provided complete protection against intravaginal (IVAG) HSV-2 challenge. There was no detectable IFN-β in mice treated with ligands for TLR4 or TLR2, and these mice were not protected against subsequent IVAG HSV-2 challenge. There was no correlation between levels of TNF-α or IFN-γ in the genital tract and protection against IVAG HSV-2 challenge following TLR ligand delivery. Both TNF-α−/− and IFN-γ−/− mice were protected against IVAG HSV-2 challenge following local delivery of poly(I:C). To confirm that type I interferon, particularly IFN-β, mediates innate protection, mice unresponsive to type I interferons (IFN-α/βR−/− mice) and mice lacking IFN regulatory factor-3 (IRF-3−/− mice) were treated with poly(I:C) and then challenged with IVAG HSV-2. There was no protection against HSV-2 infection following poly(I:C) treatment of IFN-α/βR−/− or IRF-3−/− mice. Local delivery of murine recombinant IFN-β protected C57BL/6 and IRF-3−/− mice against IVAG HSV-2 challenge. Results from these in vivo studies clearly suggest a strong correlation between IFN-β production and innate antiviral immunity against HSV-2.