Urate oxidase (Uox)-deficient mice could be an optimal animal model to study hyperuricemia and associated disorders. We develop a liver-specific conditional knockout Uox-deficient (Uox CKO) mouse using the Cre/loxP gene targeting system. These Uox CKO mice spontaneously developed hyperuricemia with accumulated serum urate metabolites. Blocking urate degradation, the Uox CKO mice showed significant de novo purine biosynthesis (DNPB) in the liver along with amidophosphoribosyltransferase (Ppat). Pegloticase and allopurinol reversed the elevated serum urate (SU) levels in Uox CKO mice and suppressed the Ppat up-regulation. Although urate nephropathy occurred in 30-week-old Uox CKO mice, 90 % of Uox-deficient mice had a normal lifespan without pronounced urate transport abnormality. Thus, Uox CKO mice are a stable model of human hyperuricemia. Activated DNPB in the Uox CKO mice provides new insights into hyperuricemia, suggesting increased SU influences purine synthesis.