T cell immunoglobulin and mucin-4 (TIM-4), mainly expressed on antigen presenting cells, plays a versatile role in immunoregulation. CD1d-restricted invariant natural killer T (iNKT) cells are potent cells involved in the diverse immune responses. It was recently reported that recombinant TIM-4 (rTIM-4) alone enhanced cytokine production in NKT hybridoma, DN32. D3 cells. Hence, we hypothesized that TIM-4 might regulate iNKT cell biology, especially their function of cytokine secretion. For the first time, we identified that TIM-4 was expressed in thymus iNKT cells, and its expression increased upon iNKT cell migration to the secondary lymphoid organs, especially in lymph nodes. Using TIM-4-deficient mice, we found that lack of TIM-4 did not disturb iNKT cell development, maturation, peripheral homeostasis and cytokine secretion. Moreover, TIM-4 deficiency did not alter the polarization of iNKT sublineages, including NKT1, NKT2 and NKT17. Finally, the mixed bone marrow transfer experiments further confirmed normal iNKT cell development and function from TIM-4-deficient bone marrow. In conclusion, our data suggest that TIM-4 is expressed on iNKT cells but dispensable for their development and function.