Skeletal muscle is the most abundant and a highly plastic tissue of the mammals, especially when it comes to regenerate after trauma, but there is limited information about the mechanism of muscle repair and its regeneration. In the present study, we found that miR‐204 is downregulated after skeletal muscle injury. In vitro experiments showed that over‐expression of miR‐204 by transfecting with miR‐204 mimics suppressed C2C12 cell proliferation, migration, and blocked subsequent differentiation, whereas inhibition of miR‐204 by transfecting with miR‐204 inhibitor showed the converse effects. Furthermore, through the dual luciferase reporter system, we demonstrated that miR‐204 can target the 3’UTR regions of Pax7, IGF1, and Mef2c and inhibit their expression. Taken together, our results suggest that Pax7, IGF1, and Mef2c are the target genes of miR‐204 in the process of myoblasts proliferation, cell migration, and differentiation, respectively, and may contribute to mouse skeletal muscle regeneration. Our results may provide new ideas and references for the skeletal muscle study and may also provide therapeutic strategies of skeletal muscle injury.