Natural killer T (NKT) cells recognize glycolipid antigens presented by the MHC class I-related glycoprotein CD1d. The in vivo dynamics of the NKT cell population in response to glycolipid activation remain poorly understood. Here, we show that a single administration of the synthetic glycolipid alpha-galactosylceramide (alpha-GalCer) induces long-term NKT cell unresponsiveness in mice. NKT cells failed to proliferate and produce IFN-gamma upon alpha-GalCer restimulation but retained the capacity to produce IL-4. Consequently, we found that activation of anergic NKT cells with alpha-GalCer exacerbated, rather than prevented, B16 metastasis formation, but that these cells retained their capacity to protect mice against experimental autoimmune encephalomyelitis. NKT cell anergy was induced in a thymus-independent manner and maintained in an NKT cell-autonomous manner. The anergic state could be broken by IL-2 and by stimuli that bypass proximal TCR signaling events. Collectively, the kinetics of initial NKT cell activation, expansion, and induction of anergy in response to alpha-GalCer administration resemble the responses of conventional T cells to strong stimuli such as superantigens. Our findings have important implications for the development of NKT cell-based vaccines and immunotherapies.