In the present study, we examined whether lipid infusion-induced insulin resistance in skeletal muscle could be reversed by the antioxidants tempol, glutathione (GSH), or tempol with GSH in male C57BL/6J mice via hyperinsulinemic-euglycemic clamp. Lipid infusion increased the mRNA level of mitochondrial type superoxide dismutase (Mn-SOD), glutathione peroxidase 1, tumor necrosis factor-alpha, and interleukin-6. Lipid infusion decreased GSH and GSH/glutathione disulfide (GSSG) ratio and increased the activities of JNK and p38 in skeletal muscle. Lipid infusion induced insulin resistance in whole body and skeletal muscle. Treatment with the SOD mimetic tempol did not prevent oxidative stress, the inflammatory response, and insulin resistance induced by lipid infusion. Tempol alone increased oxidative stress and aggravated the lipid-induced inflammatory response. GSH at 100 and 200 micromol. kg(-1) . h(-1) did not prevent insulin resistance and the inflammatory response by lipid infusion. On the contrary, GSH at 100 micromol. kg(-1) . h(-1) with tempol prevented insulin resistance in the whole body and skeletal muscle, and it completely reversed oxidative stress and the inflammatory response. These results suggest that lipid infusion-induced insulin resistance in skeletal muscle is produced by oxidative stress and cotreatment with tempol and GSH inhibits lipid-induced insulin resistance.