We previously reported that sterol-regulatory-element-binding-protein-1c (SREBP-1c) mediates insulin upregulation of genes encoding glycolytic and lipogenic enzymes in rat skeletal muscle. Here, we assessed whether glucose could regulate gene expression in contracting myotubes deriving from cultured muscle satellite cells. Glucose uptake increased twofold after a 30 minute treatment with a high glucose concentration, suggesting an acute glucose-stimulated glucose uptake. Time-course experiments showed that, within 3 hours, glucose stimulated the expression of hexokinase II, fatty acid synthase and acetyl-CoA-carboxylase-2 proteins, leading to an increased lipogenic flux and intracellular lipid accumulation in contracting myotubes. Furthermore, kinetic experiments indicated that glucose upregulated SREBP-1c precursor and nuclear proteins within 30 minutes, SREBP-1c nuclear translocation being confirmed using immunocytochemistry. In addition, the knockdown of SREBP-1 mRNA using a RNA-interference technique totally abrogated the glucose-induced upregulation of lipogenic enzymes, indicating that SREBP-1c mediates the action of glucose on these genes in rat skeletal muscle. Finally, we found that glucose rapidly stimulated SREBP-1c maturation through a Jak/STAT dependent pathway. We propose that increased intramuscular lipid accumulation associated with muscle insulin resistance in obesity or type-2 diabetes could arise partly from de novo fatty acid synthesis in skeletal muscle.