Inflammatory bowel disease (IBD) is often accompanied by metabolic imbalance and Berberine can relieve the symptoms of IBD, but the mechanism is still unclear. To explore the relationship between IBD, metabolism and Berberine, dextran sulfate sodium-induced ulcerative colitis (UC) model was built and urine and feces samples were analyzed with ultra-performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry, followed by multivariate statistical analyses. Targeted metabolomics was applied to verify and supplement the result of amino acids tested by non-targeted metabolomics. The study found that Berberine could ameliorate UC and improve metabolic disorders. The level of 4 metabolites increased and 35 decreased in urine and these metabolites mainly belong to amino acid, glucide, organic acid and purine. Besides, Berberine could reduce the level of 5 metabolites and raise the level of 7 metabolites in feces, which mainly belong to amino acid and lipid. Additionally, these altered metabolites were mainly related to amino acids metabolism, purine metabolism, vitamin metabolism, lipid metabolism and citrate cycle pathways. Furthermore, microbiome metabolism may be regulated by Berberine in UC. In general, this study provides a useful approach for exploring the mechanism of Berberine in the treatment of UC from the perspective of metabolomics.