The mechanism of 3,5,2′,4′-tetrahydroxychalcone on lowing urate level is still unknown. Here we investigated the effects of 3,5,2′,4′-tetrahydroxychalcone on urate levels, xanthine oxidase/xanthine dehydrogenase (XOD/XDH) activities in hypoxanthine-induced hyperuricemic mice, as well as the effects of 3,5,2′,4′-tetrahydroxychalcone on the mRNA expression levels and content of phosphoribosyl pyrophosphate synthetase (PRPS), phosphoribosyl pyrophosphate amidotransferase (PRPPAT) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Our results demonstrated that 3,5,2′,4′-tetrahydroxychalcone (1.0, 2.0, and 4.0 mg/kg) reduced the uric acid levels in serum of the hyperuricemic mice in dose- and time-dependent manners. The activities of XOD/XDH in serum and liver were also significantly inhibited by 3,5,2′,4′-tetrahydroxychalcone; In addition, 3,5,2′,4′-tetrahydroxychalcone decreased the mRNA expression of HGPRT in brain and content of PRPS and PRPPAT in liver. These findings demonstrated that 3,5,2′,4′-tetrahydroxychalcone suppresses uric acid production by affecting the critical enzymes, XOD/XDH, PRPS, PRPPAT and HGPRT in purine nucleotide metabolism.