Quercetin is a bioflavonoid that exhibits several biological functions in vitro and in vivo. Quercetin 3-O-methyl ether (Q3) is a natural product reported to have pharmaceutical activities, including antioxidative and anticancer activities. However, little is known about the mechanism by which it protects cells from oxidative stress. This study was designed to investigate the mechanisms by which Q3 protects against Cu2+-induced cytotoxicity. Exposure to Cu2+ resulted in the death of mouse liver FL83B cells, characterized by apparent apoptotic features, including DNA fragmentation and increased nuclear condensation. Q3 markedly suppressed Cu2+-induced apoptosis and mitochondrial dysfunction, characterized by reduced mitochondrial membrane potential, caspase-3 activation, and PARP cleavage, in Cu2+-exposed cells. The involvement of PI3K, Akt, Erk, FOXO3A, and Mn-superoxide dismutase (MnSOD) was shown to be critical to the survival of Q3-treated FL83B cells. The liver of both larval and adult zebrafish showed severe damage after exposure to Cu2+ at a concentration of 5μM. Hepatic damage induced by Cu2+ was reduced by cotreatment with Q3. Survival of Cu2+-exposed larval zebrafish was significantly increased by cotreatment with 15μM Q3. Our results indicated that Cu2+-induced apoptosis in FL83B cells occurred via the generation of ROS, upregulation and phosphorylation of Erk, overexpression of 14-3-3, inactivation of Akt, and the downregulation of FOXO3A and MnSOD. Hence, these results also demonstrated that Q3 plays a protective role against oxidative damage in zebrafish liver and remarked the potential of Q3 to be used as an antioxidant for hepatocytes. Display omitted ► Protective effects of Q3 on Cu2+-induced oxidative stress in vitro and in vivo. ► Cu2+ induced apoptosis in FL83B cells via ROS and the activation of Erk. ► Q3 abolishes Cu2+-induced apoptosis through the PI3K/Akt and MAPK/Erk pathway.