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Chito-oligosaccharides, the novel marine functional oligosaccharides, possess broad activities including anti-inflammatory and anti-oxidative effects. However, there are few studies about the molecular mechanisms of chito-oligosaccharides monomers against oxidative stress. In this study, the anti-oxidative effect and mechanism of Chitopentaose hydrochloride (COS-5) were explored in HaCaT cells. The results indicated that COS-5 improved cell viability and decreased the production of ROS induced by hydrogen peroxide (H2O2). COS-5 can increase the antioxidant-related enzymes including SOD and CAT and can reduce MDA production and LDH leakage in HaCaT cells. COS-5 effectively inhibited the decline of MMP and the expression of Caspase-3 protein. Moreover, COS-5 can significantly down-regulate abnormal activation of MAPKs pathway and up-regulate the Nrf2/ARE pathway. In summary, COS-5 has significant protective effect against H2O2-induced oxidative stress in HaCaT cells through regulating MAPKs and Nrf2/ARE pathways, suggesting that COS-5 may be used as a novel functional food supplement to prevent oxidative-stress-related diseases in the future.