Calycosin, Astragali Radix most prominent ingredient, has drawn more attention as a result of its ability to treat atherosclerosis (AS). However, the mechanism of action has not been fully elucidated. We investigated the effects of calycosin on bile acid (BA) metabolism and gut microbiome in ApoE–/– mice fed a high-fat diet (HFD). The data showed that the aorta of ApoE–/– mice treated with HFD showed significant atheromatous plaque formation and lipid accumulation, and the levels of total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) were significantly increased, while the levels of high-density lipoprotein cholesterol (HDL-C) were significantly decreased. Calycosin can substantially regulate lipid levels, thereby alleviating liver lipid deposition induced by atherosclerosis. In addition, 16S rRNA sequencing showed that calycosin treatment has reshaped the gut microbiota disturbed by HFD, in particular, increasing the ratio of Bacteroidetes/Firmicutes, and improving the relative abundance of Bilophila, Desulfovibrio, Bacteroides, Lactobacillus, etc. Meanwhile, targeted metabolomics analysis showed that calycosin treatment significantly modulated glycodeoxycholic acid (CDCA), taurocholic acids (TCA), lithocholic acid (LCA), deoxycholic acid (DCA), taurodeoxycholic acid (TDCA) and BA pool composition, which were associated with atherosclerotic plaque areas. In addition, calycosin treatment also down-regulated farnesoid X receptor (FXR) protein levels and up-regulated cytochrome P450 family 7 subfamily A member 1 (CYP7A1) protein levels in the hepatic. At the same time, calycosin inhibits the ileum FXR/TGR5 signaling pathway, inhibits BA reabsorption, promotes BA excretion, and reduces hepatic cholesterol accumulation by enterohepatic circulation. In addition, we found that calycosin significantly promoted the expression of hepatic ATP-binding cassette transporter A1 (ABCA1) and ABCG1 to mediate cholesterol efflux. Meanwhile, calycosin regulates gut microbiota, and Bacteroides, Alistipes, Desulfovibrio, Lactobacillus, Bilophila and Odoribacter are closely related to specific BAs. This enables us to further understand the relationship between BA metabolism and gut microbiota. Calycosin may reduce high-fat diet-induced hepatic cholesterol accumulation in ApoE–/– mice through gut microbiota and BA metabolism, and play a role in treating AS. Finally, we confirmed that calycosin-altered gut microbiota by fecal microbiota transplantation was sufficient to alleviate atherosclerosis. Taken together, our findings provide important insights into the pharmacological mechanisms underlying the efficacy of calycosin on atherosclerosis.