Purpose： To explore the in vitro antioxidant activity of high-nucleotide yeast hydrolysate. Methodology： The scavenging activity of diphenyl bitter acyl radical（DPPH）， hydroxyl free radical as well as the total reducing power were measured by chemical method. The oxidative stress model of the HepG2 cell induced by free fatty acid （FFA）was established. The effects of high nucleotide yeast hydrolysate on HepG2 cells were detected by thiazolyl blue tetrazolium bromide（MTT）. And the changes of reactive oxygen in the HepG2 cells were measured by fluorescent probe method. Commercial kits were used to detected the activities of intracellular oxidative damage markers such as malondialdehyde （MDA）， superoxide dismutase （SOD） and glutathione peroxidase （GSH-PX）. Real-time quantitative PCR was used to detect mRNA expression levels of keap1， Nrf2， HO-1 and NQO-1. The excitation and transposition of Nrf2 was dected by western-blot. Conclusion： 5~50 mg/mL high nucleotide yeast hydrolysate could scavenge oxygen free radicals in vitro in amass concentration-independt manner and had significant antioxidant capacity. 500-1 000 μg/mL high nucleotide yeast hydrolysate could significantly decrease the content of malonaldehyde （MDA） in the cells as well as the level of reactive oxygen species（ROS）； additionally and increase the activities of superoxide dismutase （SOD） and glutathione peroxidase （GSH-PX）. The expression mRNA levels of the antioxidant enzyme genes NQO1， Nrf2 and HO-1 were also significantly increased， and the expression mRNA level of keap1 was significantly decreased. High nucleotide yeast hydrolysate could increase the protein content in the endonuclear of HepG2 and decrease the protein content of Nrf2 in the endochylema， and this results could futher clarified that high nucleotide yeast hydrolysate could reduce FFA-induced HepG2 oxidation by activating the Nrf2 pathway injury effect.