Myogenic differentiation, which occurs in the process of muscle development, is a highly ordered process. Increasing evidence indicates that microRNAs (miRNAs) are important regulators in myogenic processes. In this study, we found that miR-204-5p expression gradually decreased when myoblasts were induced to differentiate. Our results suggested that miR-204-5p blunted myoblast differentiation, which was accompanied with a decreased proportion of myosin heavy chain (MyHC)-positive cells in myoblasts with augmented expression of miR-204-5p. Furthermore, overexpression of miR-204-5p significantly decreased the MyHC composition of slow-twitch fibers in myoblasts. Luciferase activity assays confirmed that miR-204-5p directly targeted the 3′-untranslated region (3′-UTR) of myocyte enhancer factor 2C (MEF2C) and estrogen-related receptor gamma (ERRγ). Small interfering RNA (siRNA) technology successfully inhibited the expression of MEF2C and ERRγ. Interference with MEF2C or ERRγ inhibited myoblast differentiation and the formation of slow-twitch fibers. Meanwhile, co-transfection of either si-MEF2C or si-ERRγ with miR-204-5p mimics resulted in a more severe attenuation of myogenic differentiation. In summary, this study demonstrates that miR-204-5p inhibits myoblast differentiation by targeting MEF2C and ERRγ. Our findings suggest that miR-204-5p is a potential regulator that could influence myogenesis.