Regulation of endothelial nitric oxide synthase (eNOS) in hepatocytes may be an important target in nonalcoholic fatty liver disease (NAFLD) development and progression to nonalcoholic steatohepatitis (NASH). In this study, we show genetic deletion and viral knockdown of hepatocyte-specific eNOS exacerbated hepatic steatosis and inflammation, decreased hepatic mitochondrial fatty acid oxidation and respiration, increased mitochondrial H2O2 emission, and impaired the hepatic mitophagic (BNIP3 and LC3II) response. Conversely, overexpressing eNOS in hepatocytes in vitro and in vivo increased hepatocyte mitochondrial respiration and attenuated Western diet–induced NASH. Moreover, patients with elevated NAFLD activity score (histology score of worsening steatosis, hepatocyte ballooning, and inflammation) exhibited reduced hepatic eNOS expression, which correlated with reduced hepatic mitochondrial fatty acid oxidation and lower hepatic protein expression of mitophagy protein BNIP3. The current study reveals an important molecular role for hepatocyte-specific eNOS as a key regulator of NAFLD/NASH susceptibility and mitochondrial quality control with direct clinical correlation to patients with NASH.