Development of mucosal adjuvants to generate immunity in the female genital tract may have important implications for the development of vaccines to counter sexually transmitted infections. alpha-Galactosylceramide (alpha-GalCer) is presented by CD1d molecule on APCs to invariant Valpha14(+) NKT (iNKT) cells, which upon activation rapidly produce large amounts of immunomodulatory cytokines, leading to activation of a variety of innate and adaptive immune cells. Here, we assessed whether alpha-GalCer could act as a mucosal adjuvant for induction of protective immunity against genital herpes. We found that intranasal immunization with HSV-2 glycoprotein D (gD) in combination with alpha-GalCer elicits strong systemic gD-specific IgG Ab response as well as lymphoproliferative response with a mixed Th1/Th2 cytokine profile in the spleen, mediastinal lymph nodes, and genital lymph nodes. Importantly, such an immunization scheme conferred complete protection against an otherwise lethal vaginal HSV-2 challenge. We could also show that intravaginal immunization with gD plus alpha-GalCer generates potent gD-specific lymphoproliferative and IFN-gamma responses in the genital lymph nodes and spleen. Furthermore, the vaginally immunized mice developed a strong systemic and mucosal IgG Ab response and protection against vaginal HSV-2 challenge. The mucosal adjuvant effect of alpha-GalCer was found to be mediated via CD1d molecule and appeared to be independent of the usage of the adaptor molecule MyD88. To our knowledge, this is the first report on the mucosal adjuvant effect of alpha-GalCer for induction of protective immunity against a sexually transmitted pathogen.