Abstract: In order to explore the protective mechanism of black bean peptide on hepatocyte injury induced by overload exercise in mice, black bean protein isolate was prepared from black bean. The peptides were prepared by enzymolysis and ultrafiltration with molecular weight ranged<3, 3~5 and 5~10 kDa. The model of liver injury induced by overload exercise in mice was established to study the effect of different molecular weight ranges on liver protection. The results showed that: After overload training, the liver function of the model group was abnormal, which showed that an increase in level of liver index and decrease in levels of serum ALT and AST. The liver function of mice fed with black bean polypeptide was improved in different degrees. Analysis of the data of liver tissue T-SOD, GSH-Px enzyme activity showed that black soybean polypeptide can reduce the level of oxidative stress by increasing the activity of antioxidant enzyme, and achieve the effect of anti-peroxidation damage, and the content of MDA was significantly decreased. In order to clarify the protective mechanism of black bean polypeptide on mouse liver cells, the serum TNF-α content and the relative expression of NF-κB were determined. The results showed that the serum TNF-α secretion and the relative expression of NF-κB were significantly decreased when the molecular weight of blackbean polypeptide was less than 3 kDa. In conclusion, black soybean polypeptide can effectively alleviate the abnormal liver function caused by overload exercise, increase the activity of antioxidant enzymes, reduce the level of oxidative stress, reduce the release of TNF-α and the expression of NF-κB, so as to prevent hepatocyte damage caused by cell membrane fission caused by peroxidation damage.