Aims: Nonalcoholic fatty liver disease (NAFLD) is a metabolic disorder with increasing prevalence over the last decade. Leakage of intestinal bacteria is one of the main causes that can drive the progression of NAFLD. The laxative drug lubiprostone has been reported to enhance gut barrier function. In the present study, we aimed to clarify effectiveness and mechanisms of lubiprostone as a therapeutic agent to ameliorate NAFLD. Main methods: C57BL/6 wild-type mice were fed with high-fat diet (HFD) to induce NAFLD. Two different dosages of lubiprostone and obetichoic acid were orally administered for five weeks. After sacrifice, liver injuries and intestinal physiology were evaluated. Key findings: Oral treatment of lubiprostone effectively attenuated features of HFD-induced NAFLD including liver weight, plasma liver injury markers, and hepatic steatosis. Bacterial burden in the liver was reduced after oral delivery of lubiprostone. Lubiprostone improved intestinal permeability through development of colonic mucus. Notably, levels of portal HDL cholesterol, a portal endotoxin neutralizer, were elevated by high-dosage treatment of lubiprostone. Significance: Our findings provide new insight that blockade of leaked bacterial endotoxin via lubiprostone treatment could be a therapeutic strategy to repress the development of NAFLD.