A critical role of oxidative stress in the development of nonalcoholic fatty liver disease (NAFLD) has been reported. To clarify the source of oxidative stress, hepatic expression of the superoxide-generating NADPH oxidase isoforms was examined in high-fat and high-cholesterol (HFC) diet-fed mice. The level of NOX1, but not of NOX2 or NOX4 mRNA was significantly elevated in the liver of mice fed HFC diet for 8 weeks. Increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 in HFC-fed wild-type mice (WT) were significantly ameliorated in mice deficient in Nox1 (Nox1-KO). Formation of nitrotyrosine adducts, a marker of peroxynitrite-induced injury, was apparent in hepatic sinusoids of HFC diet-fed WT, which was significantly suppressed in Nox1-KO. In fact, NOX1 mRNA was predominantly expressed in the fraction of liver sinusoidal endothelial cells (LSECs). In primary cultured LSECs, palmitic acid, the most abundant saturated free fatty acid in plasma, dose-dependently up-regulated NOX1 mRNA. Accordingly, increased oxidative stress mediated by NOX1/NADPH oxidase in LSECs may promote liver injury through peroxinitrite formation in the development of NAFLD.