Obesity is a global health problem strongly linked to gut microbes and their metabolites. In this study, ginsenoside Rg1 (Rg1) reduced lipid droplet size and hepatic lipid accumulation by activating uncoupling protein 1 expression in brown adipose tissue (BAT), which in turn inhibited high-fat diet (HFD)-induced weight gain in mice. Furthermore, the intestinal flora of mice was altered, the abundance of Lachnoclostridium, Streptococcus, Lactococcus, Enterococcus and Erysipelatoclostridium was upregulated, and the concentrations of fecal bile acids were altered, with cholic acid and taurocholic acid concentrations being significantly increased. In addition, the beneficial effects of Rg1 were eliminated in mice treated with a combination of antibiotics. In conclusion, these results suggest that Rg1 activates BAT to counteract obesity by regulating gut microbes and bile acid composition in HFD-fed mice.