AbstractMicroRNA‐543 (miR‐543) has been found to play a suppressive role in various human cancers in many studies, whereas the specific functions of miR‐543 in muscle development remain poorly understood. Here, we found that the expression of miR‐543 was high in skeletal muscle and increased during the differentiation of C2C12 cells. Overexpression of miR‐543 repressed C2C12 cell proliferation and promoted differentiation, while knockdown of miR‐543 expression produced the opposite results. During myogenesis, we predicted and verified that Krüppel‐like factor 6 (KLF6), a suppressor of multiple tumor cells, was a target gene of miR‐543. Then, miR‐543 was found to specifically target KLF6 and repress its expression. Besides this, knockdown of KLF6 promoted the differentiation but inhibited the proliferation of C2C12 cells. Si‐KLF6 can rescue the influence of miR‐543 inhibitor on C2C12 cell differentiation. Our results indicate a new regulatory mechanism of miR‐543 on KLF6 expression and suggest the possibility of using the miR‐543/KLF6 pathway as a potential target for studying myogenesis.