The present study investigated the cytoprotective properties of glycitein, a metabolite formed by the transformation of glycitin by intestinal microflora, against oxidative stress. Glycitein was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and thereby preventing lipid peroxidation and DNA damage. Glycitein inhibited apoptosis of Chinese hamster lung fibroblast (V79-4) cells exposed to hydrogen peroxide (H2O2) via radical scavenging activity. Glycitein abrogated the activation of c-Jun N-terminal kinase (JNK) induced by H2O2 treatment and inhibited DNA binding activity of activator protein-1 (AP-1), a downstream transcription factor of JNK. Taken together, these findings suggest that glycitein protected H2O2 induced cell death in V79-4 cells by inhibiting ROS generation and JNK activation.