Punicalagin has been demonstrated to exhibit anti-oxidant and anti-inflammatory properties, but whether and how it could impact hypercholesterolemia remains not fully explored. The aim of this study was to investigate the influence of punicalagin on hypercholesterolemia in mice and its related mechanisms. After 6 weeks’ intervention, punicalagin significantly reduced serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) levels in mice fed a high-fat high-cholesterol (HFHC) diet. Meanwhile, punicalagin supplementation lowered hepatic cholesterol level, which corresponded to the down-regulation of cholesterol synthesis genes (Fdps, Cyp51) and up-regulated bile acid synthesis genes (Cyp7a1, Cyp27a1). In addition, bile acid reabsorption was retarded in punicalagin-fed mice through down-regulating ileal apical sodium-dependent BA transporter (ASBT). Furthermore, intestinal farnesoid X receptor (FXR)-fibroblast growth factor 15 (Fgf15) pathway was inhibited while hepatic FXR-small heterodimeric partner (SHP) pathway was activated in punicalagin group. Microbiota analysis and targeted metabolomics showed that punicalagin decreased the abundance of bile-salt hydrolase (BSH)-producing bacteria (Clostridiaceae and Bifidobacteriaceae) and the ratio of primary BAs to secondary BAs. In conclusion, the cholesterol-lowering effect of punicalagin partly through down-regulating cholesterol synthesis and increasing cholesterol catabolism, which could be achieved by regulating gut microbiota, altering bile acid composition and modulating FXR signaling pathway. These findings indicate the potential application of punicalagin-related products as an alternative strategy for hypercholesterolemia prevention and mitigation.