Objective: Recent evidence suggests the gut microbiota-farnesoid X receptor (FXR) signaling axis is a potential therapeutic target for diabetes, but the underlying mechanism of this remains unclear. Here we investigated the influence of FXR activity on microbial biodiversity and the functional metagenome of the gut. Methods: Seven week old db/db mice were intraperitoneally injected with DMSO, FXR agonist (GW4064, 30 mg/kg/daily), or FXR antagonist ((Z)-Guggulsterone, 10mg/kg/daily) for 4 weeks and C57BLKS/J mice were used as a reference group. Body weight and food intake were monitored weekly. Glucose and insulin tolerance tests were performed during the last week of feeding. Gut microbiota profiles were established through shotgun metagenomic sequencing. Results: FXR activation by GW4064 tends to improve insulin and glucose tolerance and significantly reduced body weight. In bile acid synthesis, FXR agonist significantly inhibited CYP7A1, CYP7B1, CYP8B1 and CYP27A1 gene expression in the liver and induced fibroblast growth factor 15 in the intestine. FXR agonist treatment increased the gut microbial diversity and shifted the microbiome compositions in the feces of db/db mice, which were enriched with Lachnospiraceae and Verrucomicrobiaceae, families with beneficial health effects of fermenting short-chain fatty acids and the maintenance of mucin integrity, and depleted with the Clostridium scindens, a toxic secondary bile acid producer. Fecal microbiota transplantation from FXR agonist-treated donors to antibiotic treatment of mice also induced weight loss but did not improve glucose tolerance. Conclusion: These data showed that FXR activation by GW4064 has an anti-obesity effect in the diabetic db/db mouse, and also changes the components of gut microbiota, especially decreasing the abundance of Clostridium scindens and increasing abundance of Lachnospiraceae.