ETHNOPHARMACOLOGICAL RELEVANCE: Inonotus obliquus has also been used as a traditional folk medicine in Europe and Northeastern China to treat metabolic diseases. Betulinic acid (BA) is a major ingredient with anti-diabetic property derived from I. obliquus, however, its bioavailability is limited. Whether the beneficial effects of BA on type 2 diabetic mellitus (T2DM) referring to modulation of gut microbiota and associated metabolites remain unclear. AIM OF THE STUDY: This work aims to investigate the alleviating effect of BA on T2DM in db/db mice and elucidate the mechanism from perspective of network pharmacology, gut microbiome and fecal metabolome. MATERIALS AND METHODS: BA was orally administered to db/db mice for 45 days, and the related biochemical parameters were evaluated. The associated mechanism was explored using network pharmacology analysis, 16S rRNA sequencing and UHPLC-MS metabolomics comprehensively. Additionally, Spearman analysis was performed to assess the correlation between gut microbes, metabolites, and T2DM-related biochemical parameters. RESULTS: BA ameliorated T2DM symptoms by reducing body weight gain, regulating serum glucose and lipid levels, and mitigating T2DM-associated liver injury in db/db mice. Network pharmacology analysis indicated the ameliorative effect was via targeting at PPAR activity. BA intervention increased the relative abundance of short-chain fatty acids (SCFAs) producing bacteria including Lactobacillus and Eubacterium_xylanophilum group, and enhanced the production of SCFAs. Moreover, BA primarily regulates arginine and proline metabolism, D-glutamine and D-glutamate metabolism, and alanine, aspartate and glutamate metabolism. Spearman analysis indicated a negative correlation between SCFAs-producing bacteria and amino acids, as well as serum glucose and lipid levels. CONCLUSION: Apart from PPAR signaling pathway, BA modulated gut microbiota composition and associated metabolites in db/db mice. This study provided novel insights into the therapeutic potential of BA for alleviating T2DM symptoms.