A number of studies have shown that the natural killer T cell (NKT) ligand α-galactosylceramide (α-GalCer) serves as an adjuvant for various vaccines, including viral vaccines, parasite vaccines and protein vaccines. In this report, we investigated the adjuvant activity of α-GalCer on HIV-1 DNA vaccines in mice. This is a first study to show that α-GalCer can enhance the immunogenicity of DNA vaccines, since co-administration of α-GalCer with suboptimal doses of DNA vaccines greatly enhanced antigen-specific CD4+ T-cell and CD8+ T-cell responses. Differently from other vaccines, α-GalCer was also able to enhance HIV-specific antibody response 10-fold. It is of practical importance to find out that, in a DNA prime-DNA boost regimen, the adjuvant activity of α-GalCer was most profound when co-administered at the priming, but not at the boosting phase. In a dose-sparing experiment, we found that the level of cell-mediated immune responses in mice vaccinated with 5μg of DNA in the presence of α-GalCer was equivalent to that of mice vaccinated with 50μg of DNA in the absence of α-GalCer. Finally, results from CD1d and interferon-γ receptor knockout mice confirm our previous data and determine the mechanistic dependence upon these molecules. These results illustrate that α-GalCer enhances the immunogenicity of DNA vaccines in a mechanism-based fashion. Since both mice and humans share the CD1d molecule, this information may aid in designing more effective DNA vaccines and vaccine adjuvants against HIV-1.