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.