Nonalcoholic fatty liver disease (NAFLD), characterized by excessive fat deposition in the liver unrelated to alcohol consumption, is highly prevalent worldwide. However, effective therapeutic agents approved for NAFLD treatment are lacking. An ileal bile acid transporter inhibitor (IBATi), which represents a new mode of treatment of chronic idiopathic constipation, leads to increased delivery of bile acids to the colon. We investigated the effect of IBATi against NAFLD through modification of the gut microbiota in mice. IBATi treatment significantly suppressed body weight gain, liver dysfunction, and serum low-density lipoprotein levels and significantly decreased NAFLD activity scores in high-fat diet (HFD) mice. Treatment with IBATi ameliorated the decreased hepatic cholesterol 7-a-monooxygenase () and increased ileal fibroblast growth factor 15 () mRNA expression in HFD mice. Further, IBATi treatment changed the α-diversity in the gut microbiota reduced by HFD, which was analyzed in feces using 16S rRNA sequencing. To establish the mechanism underlying improvement in NAFLD induced by IBATi, we recolonized antibiotic solution-treated mice by fecal microbiome transplantation (FMT) using stool from HFD or HFD plus IBATi mice. This is the first report that fecally transplanted gut microbiota from HFD plus IBATi mice prevented hepatic steatosis caused by HFD. In conclusion, IBATi improved hepatic steatosis by ameliorating gut microbiota dysbiosis in NAFLD model mice, suggesting a potential therapeutic agent for NAFLD treatment. NAFLD is an increasingly recognized condition that may progress to liver cirrhosis and hepatocellular carcinoma, and community surveys have assessed that the prevalence is 14 to 32% worldwide. The first line of treatment for NAFLD is lifestyle modification to achieve weight reduction, particularly through diet and exercise. However, weight reduction is difficult to achieve and maintain, and pharmacological agents approved for the treatment of NAFLD are lacking. This study investigated the influence of the gut microbiota and the effect of an IBATi on NAFLD using a murine model. Treatment with IBATi significantly improved NAFLD in HFD mice. Further, fecal microbiome transplantation using stool from HFD plus IBATi mice prevented hepatic steatosis caused by HFD. Our study makes a significant contribution to the literature because the study findings suggest a potential treatment strategy for NAFLD patients by ameliorating gut microbiota dysbiosis.