Oxidative stress plays an important role in the pathogenesis of various liver diseases. Safflower yellow B (SYB) has been reported to protect the brain against damage induced by oxidative stress; however, whether SYB can also protect hepatocytes from oxidative stress remains unknown. In the present study, to determine whether pre-treatment with SYB reduces hydrogen peroxide (H2O2)‑induced oxidative stress in HepG2 cells, we investigated H2O2-induced oxidative damage to HepG2 cells treated with or without SYB. Cell viability was measured by MTT assay and cytotoxicity was evaluated by lactate dehydrogenase (LDH) assay. The activities of the antioxidant enzymes, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were determined using respective kits. Intracellular reactive oxygen species (ROS) accumulation in the HepG2 cells was monitored using the fluorescent marker, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). Cell apoptosis was evaluated by determining the activity of caspase-3 and by Annexin V/propidium iodide (PI) double staining. Protein expression levels were measured by western blot analysis, and the levels of related cellular kinases were also determined. H2O2 induced pronounced injury to the HepG2 cells, as evidenced by increased levels of malondialdehyde (MDA) and ROS, the decreased activity of SOD and GSH-Px, the increased activitation of caspase-3 and cell apoptosis, and the loss of mitochondrial membrane potential. SYB significantly inhibited the damaging effects of H2O2, indicating that it protected the cells against H2O2-induced oxidative damage. Moreover, pre-treatment with SYB increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and NAD(P)H dehydrogenase, quinone 1 (NQO1) which are peroxiredoxins. SYB also significantly increased the phosphorylation of AKT. However, this inductive effect was blunted in the presence of the AKT inhibitor, LY294002. The findings of our study suggest that the activation of the AKT/Nrf2 pathway is involved in the cytoprotective effects of SYB against oxidative stress. Our findings provide new insight into the cytoprotective effects of SYB and the possible mechanisms underlying these effects. Thus, SYB may prove to be of therapeutic value for the treatment of various liver diseases.