Hyperuricemia represents a significant and escalating global public health issue. Mulberry leaves, a well-established medicinal and edible plant, have historically been used in traditional diabetes treatments, though their mechanisms of action against hyperuricemia remain inadequately characterized. This study employed network pharmacology to predict the bioactive compounds and core targets of mulberry leaf extract (MLE) and validated its effects using a hyperuricemic rat model. By integrating metabolomics, molecular docking, and 16S rRNA sequencing, the study systematically investigated the regulatory effect of MLE on uric acid metabolism, endogenous pathways, and gut microbiota. The network pharmacology analysis revealed a multi-component, multi-target mechanism underlying MLE’s anti-hyperuricemic activity. Experimental findings demonstrated that MLE significantly reduced serum uric acid levels, enhanced renal urate excretion, alleviated hyperlipidemia and renal inflammation, and improved hepatic antioxidant capacity. Mechanistic studies indicated that MLE inhibited hepatic xanthine oxidase (XOD) activity and downregulated glucose transporter 9 (GLUT9) expression in the kidney and ileum, effectively modulating uric acid metabolism. Additionally, metabolomic analysis highlighted MLE’s ability to mitigate hyperuricemia-induced lipid metabolic disturbances, with xanthine dehydrogenase (XDH)/XOD identified as key therapeutic targets. Molecular docking validated the strong binding affinities of 9 major mulberry leaf constituents to XDH/XOD proteins. Furthermore, 16S rRNA sequencing revealed that MLE corrected gut dysbiosis by increasing the abundance of butyrate-producing bacteria, including Lachnospiraceae NK4A136 and Oscillospiraceae UCG-005. MetOrigin analysis and correlation studies further underscored the critical interplay between host and microbial co-metabolism in hyperuricemia. These findings collectively highlight MLE’s potential as a natural therapeutic agent for hyperuricemia, offering a robust scientific foundation for its clinical application.