Adaptations in the oxidative capacity of skeletal muscle cells can occur under several physiological or pathological conditions. We investigated the effect of increasing extracellular glucose concentration on the expression of markers of energy metabolism in primary skeletal muscle cells and the C2C12 muscle cell line. Growth of myotubes in 25 mM glucose (high glucose, HG) compared with 5.55 mM led to increases in the promoter activity, mRNA expression and enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a marker of glycolytic energy metabolism. Levels of oxidative markers peroxisome proliferator-activated receptor g coactivator 1a, citrate synthase and mitochondrial acetyl-coenzyme A acyltransferase 2 decreased. The metabolic adaptations induced by HG are comparable to those seen during a slow-to-fast fiber transformation. In addition, HG increased fast myosin heavy chain (MHC) IId/x but did not change slow MHCI/b promoter activity and mRNA expression. Protein phosphatase 2A (PP2A) was shown to mediate the effects of HG on GAPDH and MHCIId/x. The glucose-induced increase in PP2A activity was associated with increased p38 mitogen-activated protein kinase activity, which has previously been shown to be crucial for MHCIId/x gene expression. Carbohydrate response element-binding protein (ChREBP), a glucose-dependent transcription factor downstream of PP2A, partially mediated the effects of glucose on metabolic markers. Taken together, HG induces a partial slow-to-fast transformation comprising metabolic enzymes together with an increased expression of MHCIId/x. Hence, this work highlights the importance of glucose as a signaling molecule in muscle, and furthermore demonstrates a functional role for ChREBP in determining the metabolic type of muscle fibers.