OBJECTIVE The present study was undertaken to assess the contribution of inducible nitric oxide (NO) synthase (iNOS) to lipid-induced insulin resistance in vivo. METHODS Wild-type and iNOS(-/-) mice were infused for 6 h with a 20% intralipid emulsion, during which a hyperinsulinemic-euglycemic clamp was performed. RESULTS In wild-type mice, lipid infusion led to elevated basal hepatic glucose production and marked insulin resistance as revealed by impaired suppression of liver glucose production and reduced peripheral glucose disposal (R(d)) during insulin infusion. Liver insulin resistance was associated with a robust induction of hepatic iNOS, reduced tyrosine phosphorylation of insulin receptor (IR) beta, insulin receptor substrate (IRS)-1, and IRS-2 but elevated serine phosphorylation of IRS proteins as well as decreased Akt activation. The expression of gluconeogenic enzymes Pepck and G6Pc was also increased in the liver of wild-type mice. In contrast to their wild-type counterparts, iNOS(-/-) mice were protected from lipid-induced hepatic and peripheral insulin resistance. Moreover, neither the phosphorylation of insulin signaling intermediates nor expression of gluconeogenic enzymes were altered in the lipid-infused iNOS(-/-) mice compared with their saline-infused controls. Importantly, lipid infusion induced tyrosine nitration of IRbeta, IRS-1, IRS-2, and Akt in wild-type mice but not in iNOS(-/-) animals. Furthermore, tyrosine nitration of hepatic Akt by the NO derivative peroxynitrite blunted insulin-induced Akt tyrosine phosphorylation and kinase activity. CONCLUSIONS These findings demonstrate that iNOS induction is a novel mechanism by which circulating lipids inhibit hepatic insulin action. Our results further suggest that iNOS may cause hepatic insulin resistance through tyrosine nitration of key insulin signaling proteins.