--- - |2- Abstract MicroRNAs (miRNAs or miRs) are small noncoding RNAs that play critical roles in muscle cell proliferation and differentiation via post‐transcriptional regulation of gene expression. Here, based on our previous high‐throughput sequencing results, we evaluated miRNA‐499 (miR‐499) functions during myoblast proliferation and differentiation. In addition, we analyzed miR‐499 expression profiles and characterized the associated functional roles. MiR‐499 is known to be a skeletal muscle fiber‐type‐associated miRNA. However, its roles in skeletal myoblast proliferation and differentiation are poorly understood. MiR‐499 overexpression promoted C2C12 cell proliferation and significantly attenuated C2C12 cell myogenic differentiation. Furthermore, miR‐499 inhibition enhanced C2C12 cell proliferation and suppressed C2C12 cell differentiation. Using dual‐luciferase reporter assays and western blot analysis, we confirmed that miR‐499 targeted transforming growth factor β receptor 1 (TGFβR1), a known regulator of skeletal myoblast development. Additionally, our RNA interference analysis, in which TGFβR1 was downregulated, showed that TGFβR1 significantly promoted the differentiation of C2C12 cells and inhibited their proliferation. - Journal of Cellular Physiology, EarlyView.