The T cell Ig domain and mucin domain (TIM) 1 protein expressed on the surface of Th2 cells regulates the immune response by modulating cytokine production. However, the functional roles of TIM1 have not been examined in NKT cells. Therefore, we investigated the immunologic effects of TIM1 on NKT cells. We found that mouse NK1. 1+ TCR-β+, α-galactosyl ceramide/CD1d dimer+ NKT, and NKT hybridoma (DN32. D3) cells constitutively express TIM1 and TIM4 on their surface. Engagement of TIM1 on NKT cells by any of several anti-TIM1 mAbs suppressed the production of IFN-γ in the presence of TCR stimulation in vitro and in vivo, whereas the effects of such engagement on Th2 cytokine production by the NKT cells varied with the particular anti-TIM1 Ab clone. Moreover, in DN32. D3 TIM4-knockdown NKT hybridoma cells, TIM1 engagement by rTIM1 or TIM4 enhanced IL-4 production while inhibiting IFN-γ production in the presence of α-galactosyl ceramide stimulation. TIM1 engagement increased GATA-3 expression but reduced T-bet expression in NKT cells in the presence of TCR engagement. The adoptive transfer of NKT cells preincubated with anti-TIM1 mAbs into Jα18−/− mice aggravated bleomycin-induced pulmonary fibrosis by suppressing IFN-γ production. Taken together, these results suggest that TIM1 costimulation on NKT cells enhances the cellular production of IL-4 while inhibiting the production of IFN-γ. Thus, as a differential regulator of the immune response, TIM1 on NKT cells may be a useful therapeutic target for immune diseases.