Abstract Although the estimated prevalence of NAFLD is approximately 25%, an effective treatment has not been established. Recently, gut microbiota has been shown to affect the pathogenesis of NAFLD. In this study, the protective effect of Apical Sodium-dependent Bile acid Transporter inhibitor (ASBTi) against NAFLD was investigated in mice fed a high-fat diet for 12 weeks (HFD mice) via changing their gut microbiota. ASBTi mice were mice treated with ASBTi (2.5 μmol/kg/day) during HFD feeding. For fecal microbiota transplantation (FMT), stools were collected from HFD mice and ASBTi mice. Fecal materials obtained from HFD mice and ASBTi mice were transplanted to normal mice, fed HFD for 12 weeks, and designated as HFD-FMT mice and ASBTi-FMT mice, respectively. Microbiota in these groups of mice were analyzed by 16S rRNA gene sequencing. The degrees of hepatic steatosis and inflammation in these mice were determined pathologically. The shannon index of the microbiota from ASBTi mice (3.31±0.19) and ASBTi-FMT mice (3.29±0.18) was significantly high as compared to that from HFD mice (2.90±0.19) and HFD-FMT mice (2.89±0.16). Steatosis and inflammation in the liver of HFD mice and HFD-FMT mice were shown to be pathologically severe and NAFLD Activity Score (NAS) was 7.67±0.58 in HFD mice and 5.67±2.08 in HFD-FMT mice. However, those in the liver of ASBTi mice and ASBTi-FMT mice were improved and NAS was reduced in ASBTi mice (1.67±0.58) and in ASBTi-FMT mice (2.67±0.58). These results suggest that changes in the gut microbiota caused by ASBTi suppress intrahepatic steatosis and inflammation in NAFLD mice. Elobixibat can be expected to prevent fatty liver.