Repeated injection of α-galactosylceramide, an agonistic ligand for natural killer T (NKT) cells, results in long-term unresponsiveness or anergy, which severely limits its clinical application. However, the molecular mechanisms leading to NKT anergy induction remain unclear. We show here that the decreased IFN-γ production and failed tumor rejection observed in anergized NKT cells are rescued by Cbl-b deficiency. Cbl-b E3 ligase activity is critical for the anergy induction, as revealed by the similarity between Cbl-b−/− and its RING finger mutant NKT cells. Cbl-b binds and promotes monoubiquitination to CARMA1, a critical signaling molecule in NFκB activation. Ubiquitin conjugation to CARMA1 disrupts its complex formation with Bcl10 without affecting its protein stability. In addition, CARMA1−/− NKT cells are defective in IFN-γ production. The study identifies an important signaling pathway linking Cbl-b-induced monoubiquitination to NFκB activation in NKT cell anergy induction, which may help design approaches for human cancer therapy.