Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin resistant states. In muscles, glucose uptake is performed by the GLUT4 protein, which is encoded by the SLC2A4 gene. SLC2A4 gene expression increases in response to conditions that improve insulin-sensitivity, including chronic exercise. However, since chronic exercise improves insulin sensitivity, the increased SLC2A4 gene expression could not be clearly attributed to the muscle contractile activity per se, and/or to the improved insulin sensitivity. The present study was designed to investigate the role of contractile activity per se in the regulation of SLC2A4 gene expression as well as in the participation of the transcriptional factors MEF2D, HIF1a and TR. The performed in vitro protocol excluded the interference of metabolic, hormonal and neural effects. The results showed that, in response to 10 min of electrically-induced contraction of soleus muscle, an early 40% increase in GLUT4 mRNA (30 min) occurred, with a subsequent 65% increase (120 min) in GLUT4 protein content. Electrophoretic mobility shift (EMSA) and supershift assays revealed that the stimulus rapidly increased the binding activity of MEF2D, HIF1a and TR into the SLC2A4 gene promoter. Furthermore, Chromatin Immuno-Precipitation (ChIP) assay confirmed, in native nucleosome, that contraction induced a ~4-fold (P<0.01) increase in MEF2D and HIF1a binding activity. In conclusion, muscle contraction per se enhances SLC2A4 gene expression, and that involves MEF2D, HIF1a and TR transcription factors activation. This finding reinforces the importance of physical activity to improve glycemic homeostasis independently of other additional insulin sensitizer approaches.