Invariant NKT (iNKT) cells were shown to prevent the onset of experimental autoimmune encephalomyelitis in mice following administration of their specific TCR agonist α-galactosylceramide. We found that this protection was associated with the emergence of a Foxp3+ iNKT cell population in cervical lymph nodes. We demonstrate that the differentiation of these cells is critically dependent on TGF-β in both mice and humans. Moreover, in vivo generation of Foxp3+ iNKT cells was observed in the TGF-β–rich environment of the murine gut. Foxp3+ iNKT cells displayed a phenotype similar to that of Foxp3+ regulatory T cells, and they suppress through a contact-dependent, glucocorticoid-induced TNFR-mediated mechanism. Nevertheless, Foxp3+ iNKT cells retain distinctive NKT cell characteristics, such as promyelocytic leukemia zinc finger protein expression and preferential homing to the liver following adoptive transfer, where they stably maintained Foxp3 expression. Our data thus unveil an unexpected capacity of iNKT cells to acquire regulatory functions that may contribute to the establishment of immunological tolerance.