Background: Polydatin has significant uricosuric effects. This study was conducted to examine the xanthine oxidase inhibition and to detect protein levels of urate transporter 1 (URAT1), organic anion transporters 1 (OAT1), and organic anion transporters 3 (OAT3) in the hyperuricemic mice to understand the role of antihyperuricemic mechanism of polydatin. Materials and Methods: Hyperuricemia mice model was established with uricase inhibitor (potassium oxonate), and uric acids in serum were observed. Kidney tissues were used to detect gene contents of URAT1, OAT1, and OAT3 by real-time-PCR (polymerase chain reaction) and to detect pathological features. The activity of xanthine oxidase (XOD) in the liver tissues of mice was detected. Results: The polydatin experimental groups and the positive control group (benzbromarone) showed significantly inhibited levels of serum uric acid when compared with the model group. Polydatin significantly inhibited the increasing tendency of the mRNA and the protein levels of OAT1 and OAT3, and decreased the tendency of the mRNA and the protein level of URAT1. Polydatin significantly inhibited the level of XOD in the liver tissues of mice in a concentration-dependent manner. Polydatin showed a protective effect on the pathological injury of the kidney. Conclusion: Polydatin has an antihyperuricemic effect in oxonate-induced hyperuricemic mice. The effect was related to the downregulation of renal URAT1, upregulation of renal OAT1 and OAT3, and inhibition of XOD in the hyperuricemic mice.