Myogenesis, the formation and regeneration of muscular tissue, is a fundamental factor in embryonic development. N-methyl D-aspartate (NMDA) receptor synaptonuclear signaling and neuronal migration factor (Nsmf) mediates NMDA receptor endocytosis in GnRH neuronal cells. NMDA receptor is involved in myoblast differentiation by regulating Ca2 (+) dependent fusion of myocytes. In this study, we investigated the role of Nsmf in myoblast proliferation and differentiation. Quantitative-PCR, immunoblotting, and immunohistochemistry results showed that the Nsmf expression levels increased during both the differentiation and proliferation of myocytes. Knockdown of Nsmf in myocytes by siRNA did not affect the myocyte differentiation marker myogenin. However, flow cytometry showed that the proliferation rate of the Nsmf-knockdown cells was reduced compared to the control cells. Therefore, our results indicate that Nsmf is a novel myogenic factor that can enhance myoblast proliferation. Furthermore, Nsmf may be an important therapeutic target in diseases associated with aging, muscular dystrophy, or cachexia.