The liver contains significant numbers of invariant natural killer T (iNKT) cells, which have an invariant T cell receptor-alpha chain and are activated in a CD1d-restricted manner. We examined the role of iNKT cells in the spontaneous tolerance of the major histocompatibility antigen complex-mismatched liver allograft model using Jalpha18 knockout mice that lack iNKT cells. Liver allografts lacking iNKT cells manifested not only infiltration but also hemorrhage and necrosis with significant reduction of graft survival and much less induction of tolerance compared with wild type (WT) liver allograft. In addition, allografts lacking iNKT cells grafted into iNKT-deficient recipients result in more severe inflammation than when grafted into WT recipients, while there was no significant difference with respect to induction of tolerance and graft survival. These results demonstrated that iNKT cells, especially donor-residual iNKT cells, constitute immune regulatory cells that play an important role in induction of allograft tolerance.