Mucosal delivery of vaccines against sexually transmitted pathogens is important to elicit strong immune responses at biologically relevant sites. However, inclusion of appropriate adjuvants is essential to overcome the inherent mucosal tolerance. We present evidence in support of the effectiveness of co-administering alpha-galactosylceramide (α-GalCer) as an adjuvant with a CTL-inducing HIV envelope peptide, via either oral or intranasal route, to prime antigen-specific immune responses in multiple systemic and mucosal compartments. Contrary to the known potential of repeated parenteral dosing with α-GalCer to induce NKT cell anergy that could compromise adoptive immunity development, we have observed that two and three doses delivered by the intranasal or oral route were more efficient in priming broader antigen-specific immune responses. These results demonstrate the effectiveness of α-GalCer as adjuvant for repeated intranasal or oral administration of vaccines for protection against mucosally transmitted pathogens.