Isoorientin (ISO) is a flavonoid compound, and possesses a significant antioxidant potential. However, the effects of ISO on the oxidative damage in normal hepatocytes remain unknown. The present study investigated the protective effects of ISO on H₂O₂-induced apoptosis in buffalo rat liver (BRL-3A) cells. The results showed that H₂O₂ induced cell death in a dose-dependent manner, pretreatment with ISO significantly (P<0.01) increased the cell viability in a concentration-dependent manner, and no significant toxicity was found in ISO-treated cells. ISO notably decreased the loss of mitochondrial membrane potential (MMP) and the protein expression of Bax, cytochrome c (in the cytosol), cleaved caspase-3 and PARP, and ultimately reduced H₂O₂-induced BRL-3A cells apoptosis. Meanwhile, ISO also remarkably restrained the activation of ERK1/2, JNK and p38, and the inactivation of Akt induced by H₂O₂. Furthermore, ISO significantly reduced H₂O₂-induced reactive oxygen species (ROS) and nitric oxide (NO) production by elevating total superoxide dismutase (T-SOD) and catalase (CAT) levels, and inhibiting the expression of inducible nitric oxide synthase (iNOS). These results demonstrated for the first time that ISO is able to protect BRL-3A cells against H₂O₂-induced apoptosis by inhibiting mitochondrial dysfunction, inactivating MAPK kinases, activating Akt, and scavenging ROS and NO.