ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine, Morus alba L. root bark (MAR) has diuretic and detumescent effects, which is used in prescriptions like Niaoduqing granules for hyperuricemia treatment. However, the anti-hyperuricemic and nephroprotective activities, underlying mechanism and material basis of MAR have not been reported. AIM OF THE STUDY: This research aimed to explore the anti-hyperuricemic and nephroprotective activity and mechanism of MAR, along with the pursuit of potential xanthine oxidase (XOD) inhibitors within MAR. MATERIALS AND METHODS: XOD inhibitory assay and hyperuricemic mice model were employed to screen and estimate the active fraction of MAR. Then, active compositions were isolated and elucidated by diverse separation and spectroscopic techniques. The enzyme inhibition mechanism of the active compositions was investigated by enzyme kinetic and molecular docking. RESULTS: The ethyl acetate fraction (MAR-EA) showed the strongest inhibitory activity against XOD. In hyperuricemic mice, MAR-EA decreased serum uric acid levels by suppressing XOD activity and modulating renal uric acid transporters (URAT1, GLUT9, ABCG2). Moreover, it alleviated hyperuricemia-induced kidney damage, which may be related to inhibiting the production of inflammatory factors. Noticeably, the combination of MAR-EA with allopurinol showed a synergistic effect. Meanwhile, a Diels-Alder adduct, albanol A (1) was isolated from MAR-EA with excellent XOD inhibition activity (IC CONCLUSION: This research supports MAR and albanol A as anti-hyperuricemic drug candidates, laying a foundation for further exploration.