Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin-resistant states such as obesity and diabetes. Fasting induces insulin resistance, with reduced peripheral glucose utilization and tissue-specific changes in GLUT4 expression. Thus, the aim of this research was to evaluate, in fasted rat soleus, the effects of insulin on GLUT4 expression and the possible involvement of transcriptional factors myocite enhancer factor 2 (MEF2), hypoxia inducible factor 1-a (HIF1-a) and nuclear factor-κB (NF-κB). The content of GLUT4 protein was analyzed by western blotting. GLUT4, MEF2A, MEF2B, MEF2D, NF-κB and HIF1-a mRNAs were analyzed by reverse transcription-PCR. The binding activity of the nuclear proteins into the DNA sequences containing the binding site of MEF2 and NF-κB into the GLUT4 gene promoter was analyzed by eletrophoretic mobility shift assay (EMSA). The western blotting analysis showed a 20% decrease in GLUT4 protein content in soleus muscle from fasted rats compared with those fed ad libitum (p<0,05). On the other hand, muscles from fasted rats incubated during 180 minutes with insulin showed increase in GLUT4 protein content (72%, p<0,01), which was accompanied by an increase in GLUT4 mRNA (137%, p<0,01). Soleus from fasted rats, incubated by 180 minutes with either wortmannin or tumor necrosis factor-alpha showed a decrease in GLUT4 protein content of 37%, (p<0,05) and 41% (p<0,05), respectively. The addition of insulin on incubation reverted the GLUT4 changes induced by wortmannin and TNF-alpha. Rapamycin did not induce any significant change in GLUT4 protein content. MEF2C and HIF1-a mRNAs were not modulated by insulin. The incubation with insulin caused increase of MEF2A (76%, p<0,01) and MEF2D mRNAs (73%, p<0,01). Conversely, insulin decreased NF-κB mRNA (54%, p<0,05). EMSA analysis revealed an increase (58%, p<0,01) in the binding activity of the nuclear proteins from fasted rats soleus incubated for 180 minutes with insulin, into the DNA sequences containing the binding site of MEF2 into the GLUT4 gene promoter and a decreased (44%, p<0,01) into the binding site of NF-κB. These results showed that prolonged fasting and in vitro insulin stimulation conversely modulate GLUT4 protein content. In vitro incubation with insulin induced increase in GLUT4 protein probably via PI3k activation, and increased in GLUT4 mRNA. These effects were accompanied by increased MEF2A and MEF2D mRNAs and by increased binding activity to GLUT4 gene promoter, highlighting this transcriptional factor as an activator of GLUT4 expression triggered by insulin. Parallel, insulin decreased NF-κB mRNA content and binding activity to GLUT4 gene promoter, indicating this transcriptional factor as an inhibitor of GLUT4 expression.