Previously, we demonstrated that the expression of myogenin, a critical transcription factor for myogenesis, is negatively regulated by O-linked beta-N-acetylglucosamine (O-GlcNAc) glycosylation in mouse C2C12 cells. In this study, we found that Mef2 family proteins, especially Mef2D which is a crucial transcriptional activator of myogenin, are O-GlcNAc glycosylated. Between the two splice variants of Mef2D, Mef2D1a rather than Mef2D1b appears to drive the initiation of myogenin expression in the early stage of myogenesis. A deletion mutant analysis showed that Mef2D1a is glycosylated both in its DNA-binding and transactivation domains. A significant decrease in the glycosylation of Mef2D was observed in response to myogenic stimulus in C2C12 cells. Inhibition of the myogenesis-dependent decrease in the glycosylation of Mef2D suppressed its recruitment to the myogenin promoter. These results indicate that the expression of myogenin is regulated, at least in part, by the decreased glycosylation-dependent recruitment of Mef2D to the promoter region, and this is one of the negative regulatory mechanisms of skeletal myogenesis by O-GlcNAc glycosylation. (C) 2013 Elsevier Inc. All rights reserved.