It has been established that α‐galactosylceramide (α‐GalCer), a glycolipid, is recognized by natural killer T (NKT) cells together with the monomorphic MHC‐like antigen, CD1d, in mice and humans. In this study, we examined how NKT cells are modulated by in vivo administration of α‐GalCer in mice. When 2 μg (or more)/mouse of α‐GalCer was injected i.p., the majority of NKT cells disappeared in the liver and spleen, possibly undergoing apoptosis, on day 1. At this time, NKT cytotoxicity seen in liver lymphocytes also disappeared. In parallel with this numerical and functional change of NKT cells, there was always concomitant hepatocyte damage, as shown by histology and elevated levels of transaminases. Subsequently, the number and function of NKT cells continued to increase from day 3 to day 7. The response seen in hepatic (and splenic) NKT cells did not occur in thymic NKT cells. All these phenomena induced in the liver did not appear in NKT‐deficient mice such as β2‐microglobulin^–/– and CD1d^–/– mice. These results shed further light on the in vivo interaction between NKT cells and α‐GalCer in mice.