Abstract: This study aims to investigate the potential of dietary supplementation with wolfberry honey in improving inflammatory bowel disease. Wolfberry honey was collected from different apiaries in the widely cultivated wolfberry area of Golmud, Qinghai Province. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based widely targeted metabolomics was used to identify the metabolites in wolfberry honey. The main physicochemical indicators (proline content, glucose oxidase, catalase, total phenolic acids, total flavonoids) and antioxidant activity were measured. The effect of wolfberry honey supplementation (5 g/kg) on the gut microbiota in dextran sulfate sodium (DSS)-induced ulcerative colitis mice was assessed using 16S rRNA sequencing. The results showed that the glucose oxidase activity in wolfberry honey was 54.49 μg/g·0.5 h, proline content was 390.00 μg/g, and hydrogen peroxide content was 42.68 μmol/L. The total phenolic acid content was 108.00 mg GAE/kg, and the total flavonoid content was 42.47 mg/100 g. The iron ion reducing capacity (FRAP) was 0.58 mmol/L Fe²⁺/kg. The ability of wolfberry honey to scavenge DPPH, ABTS cation, and hydroxyl radicals increased with concentration, with the highest activity observed in scavenging ABTS cation radicals, showing an EC50 of 62.94 mg/mL. A total of 1201 metabolites were identified in wolfberry honey by UPLC-MS/MS, with lipids (16.94%) being the largest group, followed by carbohydrates and their derivatives (15.29%), flavonoids (13.64%), amino acids and derivatives (13.22%), alkaloids (10.74%), and phenolic acids (7.44%). For the first time, wolfberry spermidine-like compounds were identified in wolfberry honey, which could serve as biomarkers for authenticity. Wolfberry honey significantly increased the relative abundance of beneficial bacteria in the gut of DSS-induced colitis mice, such as norank_f__Muribaculaceae, Lactobacillus, Candidatus_Saccharimonas, and Faecalibaculum, and reduced the relative abundance of harmful bacteria like Escherichia-Shigella, thus maintaining gut ecological balance. This study provides a comprehensive analysis of the chemical composition of wolfberry honey and offers a theoretical basis for its application in the prevention and treatment of ulcerative colitis.