We examined whether deletion of inducible nitric oxide synthase (iNOS) could prevent lipid infusion-induced insulin resistance in iNOS-knockout and wild-type mice with the in vivo euglycemic-hyperinsulinemic clamp technique. Plasma NO metabolites were increased in lipid-infused wild-type mice, while they were not increased in iNOS-knockout mice. Plasma tumor necrosis factor-α levels were increased in both wild-type and iNOS-knockout by lipid-infusion. Lipid infusion reduced glucose infusion rate (GIR) and whole body glucose uptake in wild-type mice, whereas iNOS-knockout mice displayed comparable GIR and whole body glucose uptake compared with the control. In the gastrocnemius, lipid infusion decreased glucose uptake and glycolysis that were accompanied with increased phosphorylation of c-Jun N-terminal kinase and reduced phosphorylation of phosphoinositide 3-kinases and serine/threonine kinase Akt. However, lipid infusion did not affect glucose uptake or phosphorylation of these proteins in iNOS-knockout mice. The mRNA levels of inflammatory cytokines were also increased in the gastrocnemis of wild-type and iNOS-knockout mice by lipid infusion. Nitrotyrosine level in the gastrocnemius was increased in lipid-infused wild-type mice but it was not increased in iNOS-knockout mice. These results suggest that lack of iNOS prevents lipid infusion-induced skeletal muscle insulin resistance without attenuating cytokine levels.