ABSTRACTMicroRNAs (miRNAs), including miR‐1, miR‐133, and miR‐206, play a crucial role in muscle development by regulating muscle cell proliferation and differentiation. The aim of the present study was to define the effect of miR‐124 on myogenic differentiation of mesenchymal stem cells (MSCs). The expression level of miR‐124 in skeletal muscles was much lower than those in primary cultured bone marrow‐derived MSCs and the bone, fat and brain tissues obtained from C57BL/6 mice. Myogenic stimuli significantly decreased the expression levels of miR‐124 in mouse bone marrow‐derived MSCs and C2C12 cells. Forced expression of miR‐124 suppressed the expression of myogenic marker genes such as Myf5, Myod1, myogenin and myosin heavy chain and multinucleated myotube formation. Blockade of endogenous miR‐124 with a hairpin inhibitor enhanced myogenic marker gene expression and myotube formation. During myogenic differentiation of MSCs and C2C12 cells, the levels of Dlx5, a known target of miR‐124, were inversely regulated with those of miR‐124. Furthermore, overexpression of Dlx5 increased myogenic differentiation, whereas knockdown of Dlx5 using siRNA inhibited myogenesis in C2C12 cells. These results suggest that miR‐124 is a negative regulator of myogenic differentiation of MSCs and that upregulation of Dlx5 accompanied with downregulation of miR‐124 by myogenic stimuli is necessary for the proper progression of myogenic differentiation. J. Cell. Biochem. 115: 1572–1581, 2014. © 2014 Wiley Periodicals, Inc.