MicroRNAs (miRNAs) are a class of endogenous non‐coding RNAs that are critically involved in roles in various aspects of skeletal myogenesis. microRNA miR‐27a promotes myoblast proliferation by targeting myostatin, a critical inhibitor of skeletal muscle development, but its mode of action in myoblast differentiation remains unclear. We have found that expression of miR‐27a and myostatin were upregulated and downregulated, respectively, during myoblast differentiation. Overexpression of miR‐27a increased the number of myosin heavy chain (MHC)‐positive cells and upregulated mRNA and protein of MyoD and myogenin. These findings indicate that miR‐27a plays a role in enhancing myoblast differentiation.