The aim of this study is to investigate the hypouricemic effect of black rice anthocyanins in vivo and the possible mechanism of their interaction with xanthine oxidase (XO). Four different secondary purified fractions were further prepared from the crude extract of black rice anthocyanins. In vitro enzyme inhibition experiments showed that fraction 2 indicated a reversible mixed inhibition of XO and the best inhibition. Fluorescence quenching spectroscopy and circular dichroism identified a single binding site within the active cavity of XO, the dominant molecular force was van der Waals, and the binding of anthocyanins to XO altered the standing secondary structure of the protein and inhibited the active center of the enzyme. Using a mouse model of hyperuricemia, fraction 2 and crude extract of black rice anthocyanins were found to have a strong ability to reduce uric acid levels and inhibit key enzymes in the uric acid production pathway, and to reduce kidney damage and oxidative stress. This study suggests that black rice anthocyanins have potential future applications as an effective anti-hyperuricemia bioactive substance. • Xanthine oxidase (XO) is a key enzyme in the metabolism of purines. • Black rice is rich in cyanidin 3-O-glucoside (C3G), a widely known anthocyanin. • Fluorescence spectroscopy to investigate the mechanism of XO inhibition by C3G. • Circular dichroism to investigate the mechanism of XO inhibition by C3G. • C3G has a very good effect on uric acid and oxidative stress.