Reactive oxygen species (ROS) are known to play a critical role in the development of non-alcoholic steatohepatitis (NASH). To clarify the source of ROS, we examined the expression of superoxide-generating NADPH oxidase isoforms in the liver of high-fat and high-cholesterol (HFC) diet-fed mice. The mRNA expression of NOX1 was significantly elevated in mice on HFC diet for 8 weeks. Increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 in HFC diet-fed wild-type mice (WT) were significantly ameliorated in mice deficient in Nox1 (Nox1-KO). Increased nitrotyrosine adduct formation, a marker of peroxynitrite-induced injury, was observed in hepatic sinusoids of WT, which was significantly suppressed in NOX1-KO. NOX1 mRNA was mainly expressed in liver sinusoidal endothelial cells (LSECs), and it was significantly up-regulated in primary cultured LSECs treated with palmitic acid (PA). The production of nitric oxide by LSECs and LSECs-dependent relaxation of hepatic stellate cells were significantly attenuated by PA treatment. In contrast, these effects of PA were not observed in cells isolated from Nox1-KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impair hepatic microcirculation through reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the development of NASH.