Mulberry (Fructus mori) is a traditional Chinese fruit that has beneficial effects due to its numerous biological activities. This study aimed to investigate the anti-hyperuricemic activity and underlying mechanism of laboratory-prepared mulberry water extract in mice with hyperuricemia (HUA). Additionally, the effect of mulberry extract (ME) on the microbiota was investigated. The results demonstrated that ME reduced the levels of HUA-related biochemical indices [uric acid (UA), creatinine (Cr), and blood urea nitrogen (BUN)] and pro-inflammatory factors (TNF-α, IL-6, IL-8, and IL-1β) in the serum of HUA model mice. ME suppressed xanthine oxidase (XOD) and adenosine deaminase (ADA) activity while modulating the expression of the urate transporters ATP-binding cassette transporter G2 (ABCG2) and recombinant urate transporter 1 (URAT1) in the kidney. Furthermore, high-dose ME modulated the microbiota, including Ligilactobacillus, Prevotellaceae, Bacteroides and Desulfovibrio. Overall, these results demonstrate the efficacy of ME in alleviating HUA by inhibiting XOD and ADA activity, as well as modulating transport proteins to decrease urate synthesis. Additionally, ME regulates the microbiota associated with host UA metabolism. These findings confirm the UA-lowering effects of ME, highlighting its potential as a therapeutic agent for HUA.