Aims: The current study aim was to elucidate the antihypoglycemic role and mechanism of inducible nitric oxide synthase (iNOS) under inflammatory stress. Methods: Liver inflammatory stress was induced in wild-type (WT) and iNOS-knockout (iNOS-/-) mice by lipopolysaccharide (LPS) (5 mg/kg) with and without the background of nonalcoholic steatohepatitis (NASH)-Induced by high cholesterol diet (HCD, 6 weeks). Results: HCD led to steatohepatitis in WT and iNOS-/- mice. LPS administration caused marked liver inflammatory damage only in cholesterol-fed mice, which was further exacerbated in the absence of iNOS. Glucose homeostasis was significantly impaired and included fatal hypoglycemia and inhibition of glycogen decomposition. In iNOS-/- hypoxia-inducible factor-1 (HIF1), signaling was impaired compared to control WT. Using hydrodynamic gene transfer method HIF1α was expressed in the livers of iNOS-/- mice, and significantly ameliorated cholesterol and LPS-induced liver damage. WT mice overexpressing HIF1α exhibited higher blood glucose levels and lower glycogen contents after LPS injection. Conversely, induction of HIF1α was not effective in preventing LPS-induced glucose lowering effect in iNOS-/- mice. The critical role of NO signaling in hepatocytes glucose output mediated by HIF1 pathway was also confirmed in vitro. Results also demonstrated increased oxidative stress and reduced heme oxygenase-1 mRNA in the livers of iNOS -/- mice. Furthermore, the amounts of plasma tumor necrosis factor-α (TNFα) and intrahepatic TNFα mRNA were significantly elevated in the absence of iNOS. Innovation and Conclusion: These data highlight the essential role of iNOS in the glycemic response to LPS in NASH conditions and argues for the beneficial effects of iNOS. Antioxid. Redox Signal. 19, 1889-1901.