Acute kidney injury (AKI) is a common side effect of acetaminophen (APAP) overdose. Dihydromyricetin (DHM) is the most abundant flavonoid in rattan tea, which has a wide range of pharmacological effects. In the current study, APAP-induced AKI models were established both in vivo and in vitro. The results showed that DHM pretreatment remarkably alleviated APAP-induced AKI by promoting antioxidant capacity through the nuclear factor erythroid-related factor 2 (Nrf2) signaling pathway in vivo. In addition, DHM reduced ROS production and mitochondrial dysfunction, thereby alleviating APAP-induced cytotoxicity in HK-2 cells. The way in which DHM improved the antioxidant capacity of HK-2 cells was through promoting the activation of the Nrf2-mediated pathway and inhibiting the expression levels of inflammation-related proteins. Furthermore, Nrf2 siRNA partially canceled out the protective effect of DHM against the cytotoxicity caused by APAP in HK-2 cells. Altogether, the protective effect of DHM on APAP-induced nephrotoxicity was related to Nrf2-dependent antioxidant and anti-inflammatory effects.