Quercus acuta Thunb. (Fagaceae) (QA) is cultivated as a dietary and ornamental plant in China, Japan, South Korea, and Taiwan. It has been widely used as the main ingredient of acorn tofu, a traditional food in China and South Korea. The aim of this study was to determine in vitro and in vivo xanthine oxidase (XO) inhibitory and antihyperuricemic activities of an ethyl acetate extract of QA leaf (QALE) and identify its active phytochemicals using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC) systems. The QALE was found to possess potent in vitro antioxidant and XO inhibitory activities. In vivo study using hyperuricemic mice induced with potassium oxonate demonstrated that the QALE could inhibit hepatic XO activity at a relatively low oral dose (50 mg/kg) and significantly alleviate hyperuricemia to a similar extent as allopurinol. Several active compounds including vitamin E known to possess XO inhibitory activity were identified from the QALE. To the best of our knowledge, this is the first study that reports the active constituents and antihyperuricemic effect of QA, suggesting that it is feasible to use QALE as a food therapy or alternative medicine for alleviating hyperuricemia and gout.