Skeletal muscle is an essential tissue for maintaining the body's basic functions. The basic structural unit of skeletal muscle is the muscle fiber, and its type is the main factor that determines the athletic ability of animals. The O-linked N-acetylglucosamine (O-GlcNAc) modification, a reversible protein post-translational modification, is involved in many important biological processes such as gene transcription, signal transduction, cell growth, and differentiation. Myogenic differentiation factor (MyoD), the first discovered myogenic regulatory factor, facilitates the transformation of fibroblasts into skeletal muscle cells. In early laboratory studies, MyoD was found to be modified by O-GlcNAcylation. However, the regulatory effects and mechanisms of O-GlcNAcylation modification on MyoD in skeletal muscle development and differentiation remain unclear.Therefore, our research was aimed at exploring the mechanism of MyoD in skeletal muscle differentiation under the influence of O-GlcNAcylation modification, through O-linked N-acetyl glucosamine transferase (OGT) or O-N-acetylaminoglucosidase (OGA) manipulation, as well as MyoD supplementation. During the differentiation of C2C12 cells, O-GlcNAcylation of MyoD was found to be mediated by OGT, through its interaction with MyoD. Additionally, OGT was found to antagonize with UPF1 in inhibiting the ubiquitination-mediated degradation of MyoD via the K48 site, thereby regulating myotube formation. In mouse skeletal muscle tissue, Ogt gene deletion led to the differentiation of mouse skeletal muscle fibers from fast-twitch muscle fibers to slow-twitch muscle fibers, whereas this effect was mitigated by supplementation with exogenous MyoD. These results enhance understanding of the regulatory mechanisms of O-GlcNAcylation modification of MyoD in muscle development and differentiation. Our findings also indicate potential therapeutic targets for muscle and metabolism-related diseases.