The altered circulating bile acids (BAs) modulate gut microbiota, energy metabolism and various physiological functions. BA profiles in liver, serum, ileum and feces of HFD-fed mice were analyzed with normal chow diet (NCD)-fed mice after 16-week feeding. Furthermore, gut microbiota was analyzed and its correlation analysis with BA was performed. The result showed that long-term HFD feeding significantly decreased hepatic and serum BA levels, mainly attributed to the inhibition of hepatic BA synthesis and the reduced reabsorption efficiency of BAs in enterohepatic circulation. It also significantly impaired glucose and lipid homeostasis and gut microbiota in mice. We found significantly higher bile salt hydrolase activity in ileal microbes and a higher ratio of free BAs to conjugated BA content in ileal contents in HFD groups compared with NCD group mice, which might account for the activated intestinal farnesoid X receptor signaling on liver BA synthesis inhibition and reduced ileal reabsorption. The decreased circulating BAs were associated with the dysregulation of the lipid metabolism according to the decreased TGR5 signaling in the ileum and BAT. In addition, it is astonishing to find extremely high percentages of taurocholate and 12-OH BAs in liver and serum BA profiles of both groups, which was mainly attributed to the high substrate selectivity for 12-OH BAs of the intestinal BAs transporter during the ileal reabsorption of enterohepatic circulation. This study revealed a significant effect of long-term HFD feeding on the decreased circulating BA pool in mice, which impaired lipid homeostasis and gut microbiota, and collectively resulted in metabolic disorders and obesity.