The effects of mesenchymal stem cells (MSCs) on phenotype and function of natural killer T (NKT) cells, is not understood. We used concanavalin A (ConA) - and α-galactosylceramide (α-GalCer)-induced liver injury to evaluate effects of MSCs on NKT-dependent hepatotoxicity. Mouse MSCs (mMSCs) significantly reduced Con A- and α-GalCer-mediated hepatitis in C57Bl/6 mice, as demonstrated by histopathological and biochemical analysis, attenuated influx of inflammatory (T-bet(+) TNF-α, IFN-γ producing and GATA3(+) , IL-4-producing) liver NKT cells and down-regulated TNF-α, IFN-γ and IL-4 levels in the sera. The liver NKT cells cultured in vitro with mMSCs produced lower amounts of inflammatory cytokines (TNF-α, IFN-γ, IL-4) and higher amounts of immunosuppressive IL-10 upon α-GalCer stimulation. mMSC treatment attenuated expression of apoptosis-inducing ligands on liver NKT cells and suppressed the expression of pro-apoptotic genes in the livers of α-GalCer-treated mice. mMSCs reduced cytotoxicity of liver NKT cells against hepatocytes in vitro. The presence of 1-methyl-DL-tryptophan, a specific inhibitor of indoleamine 2,3-dioxygenase (IDO) or L-N(G) -monomethyl Arginine citrate, specific inhibitor of inducible nitric oxide synthase (iNOS), in mMSC-conditioned medium injected to α-GalCer-treated mice, counteracted the hepatoprotective effect of mMSCs in vivo, and restored pro-inflammatory cytokine production and cytotoxicity of NKT cells in vitro. Human MSCs in iNOS and IDO-dependent manner, attenuated the production of inflammatory cytokines in α-GalCer-stimulated human peripheral blood mononuclear cells and reduced their cytotoxicity against HepG2 cells. In conclusion, MSCs protect from acute liver injury by attenuating cytotoxicity and capacity of liver NKT cells to produce inflammatory cytokines in iNOS and IDO dependent manner.