Abstract: To investigate the anti-hyperuricemic and nephroprotective effects of barley leaf powder (BLP) in hyperuricemic mice and the underlying mechanisms, hyperuricemia in mice via intraperitoneal injection of potassium oxazinate were induced, and the effects by measuring the serum uric acid (UA), urine UA, feces UA, serum creatinine (CR), urea nitrogen (BUN), serum and hepatic xanthine oxidase (XOD) activities were evaluated, the pathological changes of the kidney tissues by HE staining were observed. Besides, the mRNA expression levels of urate transporters including urate transporter 1 (URAT1), glucose transporter 9 (GLUT9) and organic anion transporter 1 (OAT1) were detected to explore mechanisms of the anti-hyperuricemic and nephroprotective effects of BLP. Results showed that, compared with the model group, BLP at 200 mg/kg could highly significantly reduce the serum UA level (P<0.001), promote the excretion of UA from urine (P<0.001) and extremely significantly promote the UA excretion in feces (P<0.01). Meanwhile, BLP highly significantly reduced the serum and hepatic XOD activities as well as the production of liver UA at 200 mg/kg (P<0.001). In addition, compared with the model group, 200 mg/kg BLP could extremely significantly reduce the elevated serum CR (P<0.01) and highly significantly reduce the BUN levels caused by hyperuricemia (P<0.001), as proved by the pathological analysis of the kidney tissues. Data from qRT-PCR assays showed that 200 mg/kg BLP highly significantly (P<0.001) downregulated URAT1 and GLUT9 and upregulated OAT1 expressions compared with the model group. In summary, the anti-hyperuricemic and nephroprotective effects of BLP may be mediated by regulation of XOD, URAT1, GLUT9 and OAT1.