Xanthine oxidase (XOD) is a key enzyme in uric acid production, and its increased activity is closely associated with hyperuricemia and gout. Mangiferin has been shown to inhibit XOD, but the molecular mechanisms underlying this inhibition remain poorly understood. To elucidate the inhibitory effect of mangiferin on XOD and its underlying molecular mechanisms, this study employed enzyme activity assays, inhibition kinetics, fluorescence spectroscopy, thermodynamic analysis, circular dichroism (CD), and molecular docking simulations to investigate the interaction between mangiferin and XOD. Results indicated that mangiferin inhibits XOD via a reversible mixed-type inhibition mechanism in a dose-dependent manner, achieving a maximum inhibition rate of 80.05%, which was comparable to that of allopurinol (81.58%). Fluorescence and thermodynamic analyses revealed the formation of a stable complex between mangiferin and XOD, with the binding process being an endothermic spontaneous reaction driven primarily by hydrophobic interactions. Synchronous fluorescence and CD results demonstrated that mangiferin alters the microenvironment around tryptophan residues and modifies the secondary structure of XOD, thereby affecting its catalytic activity. Molecular docking further revealed that mangiferin interacted with multiple key amino acid residues through hydrogen bonding and hydrophobic interactions. This study provides a theoretical foundation for the development and application of mangiferin in functional foods targeting hyperuricemia and gout, as well as insights for designing safer and more effective XOD inhibitors.