Abstract: To investigate the effects of giant salamander active peptides (GSAP) on gut microbiota and metabolites in high-fat diet-induced obese mice, obese mouse model was established with high-fat diet. Mice in the treatment group were orally administered with GSAP for 30 days. On the final day, feces and serum samples were collected, and blood lipid biochemical indicators were measured using a reagent kit. The effects of GSAP on intestinal microbiota diversity and composition were analyzed by high-throughput sequencing technology (RNA-seq). Additionally, untargeted metabolomics was employed to evaluate the effect of GSAP on mouse fecal metabolites. Results showed that, the Lee's index of mice in the middle-dose GSAP group (M-GSAP) and high-dose GSAP group (H-GSAP) was significantly lower than those in OM (P<0.05). Serum lipid levels in all dose GSAP groups were better than those in OM. High-throughput sequencing results revealed that, intervention with GSAP restored the relative abundance of gut microbiota in OM mice to the normal control group (NC) mice. Untargeted metabolomics using UHPLC-Q-TOF MS showed some bile acids (chenodeoxycholate, trihydroxycholestanoic acid, 1,24-dihydroxyvitamin D₃, cholecalciferol, 3β-hydroxy-5-cholenoic acid, ursodeoxycholic acid, isodeoxycholic acid) and lipids (Pc (16:1e/9-hode), Pg 44:12, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, etc.) were decreased. Giant salamander active peptides alleviate gut microbiota dysbiosis in obese mice induced by high-fat diet, they hold potential as a functional food ingredient for lipid-lowering and weight loss by regulating the structure of gut microbiota.