Our recent study demonstrated that the dynamic changes of gut microbiota mediated by Flammulina velutipes polysaccharide (FVP) could effectively regulate the lipid metabolism in high fat diet-fed (HFD-fed) obese mice model. In this paper, further research was carried out by examining the bile acid (BAs) profiles, as well as the BAs metabolic pathways changes in obese mice. Furthermore, the regulatory effect of BAs on lipid metabolism was verified by 3 T3-L1 preadipocyte differentiation model. The FVP administration resulted in lower BAs content in plasma of obese mice. From the qRT-PCR analysis, FVP could relieve cholestasis in obese mice through altering the BAs metabolic pathways, changing the related genes expressions in mice liver and ileum. The cholic acid (CA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA) and ursodeoxycholic acid (UDCA) were selected in cell experiment which all reduced the intracellular triglyceride content and increased the expression of AMPKα1 in 3 T3-L1 adipocytes. Furthermore, CA and CDCA were found increased the expression of PPARα. In combination with our previous research, we further confirmed in this paper that the changes of BAs metabolism caused by FVP showed a positive effect on lipid metabolism, both in obese mice and 3 T3-L1 adipocytes.