Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are continually produced during metabolic processes. ROS generation is normally counterbalanced by the action of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and other redox molecules. However, excessive ROS will attack membrane lipids, proteins and DNA, which were believed to be involved in many health disorders such as diabetes mellitus, cancer, neurodegenerative and inflammatory diseases. On the other hand, food oxidation processes, especially lipid oxidation, lead to the formation of secondary lipid peroxidation products and degradation of food quality. Antioxidants may directly react with and quench reactive oxygen species (ROS). The utilization of synthetic antioxidants is limited because consumers are increasingly demanding additive-free or natural products. Therefore, the application of natural antioxidants as sources of many effective antioxidants is a promising alternative to the use of synthetic antioxidants. More recently, there is growing interest in natural hydrolysed protein antioxidants from many animal and plant sources, including those derived from soybean, zein, Pacific hake, algae, and smooth hound muscle. In our previous study, antioxidative peptides (1-4 ku) obtained from Scomberomorous niphonius skin (FractionⅡ) had a high antioxidant in cooked pork patties because it inhibited both lipid and protein oxidation and stabilized the quality during refrigerated storage. The purpose of the present study is to evaluate protective effects of the S. niphonius skin peptides(1-4 ku)against H₂O₂-induced oxidative stress in Caco-2 cells. There were five treatments in this experiment, including normal control (samples were not treated with H₂O₂ and antioxidant peptide), negative control, H₂O₂+Low-dose of FractionⅡ (10 μg/mL), H₂O₂+Middle-dose of FractionⅡ (50 μg/mL), H₂O₂+High-dose of FractionⅡ (100 μg/mL). The viability of cells, antioxidant enzymes activity including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), lactic dehydrogenase (LDH), and MDA content were determined. Further, the flow cytometry and fluorescence microscopy were also applied to detect the cell apoptosis. The result showed that the contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) levels were significantly increased compared with negative control group, while the lactic dehydrogenase (LDH) and MDA contents was sharply decreased treated by middle and high dose of FractionⅡ. Treatment with high dose FractionⅡ(100 μg/mL) could increase the cell viability rate to 47.10%, increase SOD, GSH-Px and CAT activity to 45.50 U/mg protein, 35.10 U/mg protein and 34.13 U/mg protein respectively, and decrease the levels of LDH activity and MDA content to 20.93 U/mg protein and 20.77 nmol/mg protein. Moreover, the flow cytometric analysis and fluorescence microscopy observation confirmed that a certain amount of FractionⅡ have a more effective role in decreasing cells apoptosis caused by oxidative stress. These results demonstrated that preincubated Caco-2 cells with antioxidant peptide (S. niphonius skin hydrolysates) resulted in a significant protection against the toxicity caused by H₂O₂.