Invariant NKT (iNKT) cells recognize glycolipid Ags, such as the marine sponge-derived glycosphingolipid α-galactosylceramide (αGalCer) presented by the CD1d protein. In vivo activation of iNKT cells with αGalCer results in robust cytokine production, followed by the acquisition of an anergic phenotype. Here we have investigated mechanisms responsible for the establishment of αGalCer-induced iNKT cell anergy. We found that αGalCer-activated iNKT cells rapidly up-regulated expression of the inhibitory costimulatory receptor programmed death (PD)-1 at their cell surface, and this increased expression was retained for at least one month. Blockade of the interaction between PD-1 and its ligands, PD-L1 and PD-L2, at the time of αGalCer treatment prevented the induction iNKT cell anergy, but was unable to reverse established iNKT cell anergy. Consistently, injection of αGalCer into PD-1-deficient mice failed to induce iNKT cell anergy. However, blockade of the PD-1/PD-L pathway failed to prevent bacterial-or sulfatide-induced iNKT cell anergy, suggesting additional mechanisms of iNKT cell tolerance. Finally, we showed that blockade of PD-1/PD-L interactions enhanced the antimetastatic activities of αGalCer. Collectively, our findings reveal a critical role for the PD-1/PD-L costimulatory pathway in the αGalCer-mediated induction of iNKT cell anergy that can be targeted for the development of immunotherapies.