Abstract: The present study was designed to explore the effects and mechanism of hyperoside on anti-fatigue in mice. The mice were randomly divided into four groups, including the control group, low-dose (5 mg/kg) hyperoside group, medium-dose (10 mg/kg) hyperoside group and high-dose (20 mg/kg) hyperoside group. The rotating rod test and exhaustive swimming test were used to determine the anti-fatigue effect of hyperoside. Then, the lactic acid (LA), blood urea nitrogen (BUN), liver glycogen (LG), muscle glycogen (MG), reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) were measured using enzyme-linked immunosorbent assay. There after, the binding energy between hyperoside and nuclear factor erythroid 2-related factor 2 (Nrf2), and the protein expressions of Nrf2, heme oxygenase 1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1) were detected by molecular docking and Western blot, respectively. The results showed that hyperoside (5, 10, 20 mg/kg) significantly increased the rotating rod lasting time and the exhaustive swimming time in mice than those of control group (P<0.05). Moreover, hyperoside markedly reduced the contents of LA and BUN, while increased the contents of LG and MG in mice than those of control group (P<0.05); hyperoside also obviously decreased the contents of ROS and MDA, increased the activities of SOD and GSH-Px in mice than those of control group (P<0.05). Of note, the binding energy of hyperoside with Nrf2 was −11.21 kcal/mol, which indicated that Nrf2 might be the potential target of hyperoside. Furthermore, hyperoside increased the level of Nrf2 in nucleus, as well as up-regulated the protein expressions of HO-1 and NQO-1. These findings suggested that hyperoside exerted anti-fatigue effect and antioxidative activity through mediating Nrf2 signaling pathway.