The aim of this study was to determine the effects of soybean protein hydrolysates against intracellular antioxidant activity. Soybean peptides (1000 to 2000 Da range) were extracted by soybean proteolysis and ultrafiltration and sequenced with a Nano-LC-ESI-MS/MS. In this study we found that soybean peptides inhibited the production of reactive oxygen species (ROS) induced by hydrogen peroxide (H2O2), malondialdehyde (MDA) and oxidized glutathione (GSSG) in HepG2 cells. Moreover, they also prevented the reduction of reduced glutathione (GSH) and up-regulated cellular resistance oxidase activity. In addition, soybean peptide treatment stimulated the mRNA and protein expression levels of antioxidant enzymes and nuclear factor erythroid-2-related factor 2 (Nrf2). Activated Nrf2 up-regulated antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)) and inhibited ROS and MDA production. It was concluded that soybean peptides effectively activated the Nrf2/antioxidant response element (ARE) mediated activity.