Dendritic cells (DCs) loaded with alpha-galactosylceramide (alpha-GalCer) are known to be active APCs for the stimulation of innate NKT and NK cell responses in vivo. In this study, we evaluated the capacity of non-DCs to present alpha-GalCer in vitro and in vivo, particularly tumor cells loaded with alpha-GalCer (tumor/Gal). Even though the tumor cells lacked expression of CD40, CD80, and CD86 costimulatory molecules, the i.v. injection of tumor/Gal resulted in IFN-gamma secretion by NKT and NK cells. These innate responses to tumor/Gal, including the induction of IL-12p70, were comparable to or better than alpha-GalCer-loaded DCs. B16 melanoma cells that were stably transduced to express higher levels of CD1d showed an increased capacity relative to wild-type B16 cells to present alpha-GalCer in vivo. Three different tumor cell lines, when loaded with alpha-GalCer, failed to establish tumors upon i.v. injection, and the mice survived for at least 6 mo. The resistance against tumor cells was independent of CD4 and CD8 T cells but dependent upon NKT and NK cells. Mice were protected from the development of metastases if the administration of live B16 tumor cells was followed 3 h or 3 days later by the injection of CD1d(high)-alpha-GalCer-loaded B16 tumor cells with or without irradiation. Taken together, these results indicate that tumor/Gal are effective APCs for innate NKT and NK cell responses, and that these innate immune responses are able to resist the establishment of metastases in vivo.