The glycolipid α ‐galactosylceramide (α ‐GalCer), which is presented by CD1d and specifically activates Vα 14 NKT cells, exerts a potent anti‐metastatic effect when administered in vivo. In this study, we demonstrated that α ‐GalCer administration led to rapid elimination of NKT cells by apoptosis in the liver and spleen, after they produced IFN‐γ  and IL‐4. In contrast, a more prolonged secretion of IFN‐γ  was observed by liver and splenic NK cells after α ‐GalCer administration. Cytotoxic activity of liver mononuclear cells was not augmented 3h after α ‐GalCer administration, but was increased at 24 h when NKT cells were mostly depleted. The α ‐GalCer‐induced cytotoxic activity was abolished in IFN‐γ ‐deficient and NK cell‐depleted mice as well as CD1‐deficient mice, suggesting that the α ‐Galcer‐induced cytotoxicity was mainly mediated by IFN‐γ ‐activated NK cells. While the α ‐GalCer‐induced cytotoxicity in vitro was mostly perforin dependent, anti‐metastatic effect of α ‐GalCer was impaired in NK cell‐depleted or IFN‐γ ‐deficient mice but not in perforin‐deficient mice. Collectively, these results indicated that the anti‐metastatic effect of α ‐GalCer is mainly mediated by NK cells, which are activated secondarily by IFN‐γ  produced by α ‐GalCer‐activated NKT cells, in aperforin‐independent manner.