Chitooligosaccharides (COS) are hydrolyzed products of chitosan and have been proven to exhibit various biological functions. The aims of this study were to investigate the mechanisms underlying the hepatoprotective effects of COS against ethanol-induced oxidative stress in vitro. Human L02 normal liver cells were pretreated with COS (0.25, 0.5 and 1.0 mg/ml) and then hepatotoxicity was stimulated by the addition of ethanol (80 mM). Pretreatment with COS protected L02 cells from ethanol-induced cell cytotoxicity through inhibition of reactive oxygen species generation. Furthermore, ethanol-induced lipid peroxidation and glutathione depletion was inhibited by COS. The antioxidant potential of COS was correlated with the induction of antioxidant genes including HO-1, NQO1 and SOD via the transcriptional activation of nuclear factor erythroid-2‑related factor-2 (Nrf2). Additionally, the protective effects of COS against ethanol were blocked by Nrf2 knockdown. Moreover, signal transduction studies showed that COS was able to suppress the ethanol-induced phosphorylation of p38 MAPK, JNK and ERK. In conclusion, the COS-mediated activation of Nrf2 and reduction of MAPK phosphorylation may be important for its hepatoprotective action.