CD1d-restricted natural killer T cell (NKT) is a subset of T cells and plays an important role in the regulation of diverse immune responses. MicroRNA-mediated RNA interference is emerging as a crucial regulatory mechanism in the control of NKT cell development and function. Yet, roles of specific microRNA in the development and function of NKT cells is not completely understood. In this study, miR-150 knockout (miR-150KO) mice were adopted and the quantities of thymic and peripheral NKT cells were detected by flow cytometry. Cytokine production was detected by intracellular staining and ELISA. We found that miR-150 deletion resulted in the decreased number of thymic NKT cells, while peripheral NKT cells did not change in mice. However, activated NKT cells in miR-150KO mice produced more IFN-γ than that of wild type control (WT) mice. In addition, using B16BL6 melanoma mouse model, we found that miR-150 deletion enhanced the inhibitory effect of α-Galcer on the lung metastasis of melanoma cells. Our data provide new clues for the specific role of miR-150 in the development and function of NKT cells.