CD1d-restricted Vα14 invariant NKT (iNKT) cells play 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 iNKT cell differentiation and function. Yet, roles of specific microRNAs in the development and function of iNKT cells remain to be further addressed. In this study, we identified the gradually increased expression of microRNA-150 (miR-150) during the maturation of iNKT cells in thymus. Using miR-150 knockout (KO) mice, we found that miR-150 deletion resulted in an interruption of iNKT cell final maturation in both thymus and periphery. Upon activation, iNKT cells from miR-150KO mice showed significantly increased IFN-γ production compared with wild-type iNKT cells. Bone marrow-transferring experiments demonstrated the cell-intrinsic characteristics of iNKT cell maturation and functional defects in mice lacking miR-150. Furthermore, miR-150 target c-Myb was significantly upregulated in miR-150KO iNKT cells, which potentially contribute to iNKT cell defects in miR-150KO mice. Our data define a specific role of miR-150 in the development and function of iNKT cells.