Our previous study showed that 3,3′-diselenodipropionic acid (DSePA), a simple, stable, and water-soluble organoselenium exhibiting glutathione peroxidise (GPx)-like activity offered good radio-protection under in vitro and in vivo conditions. Herein, we investigated the anti-genotoxic  effect of DSePA in model cellular systems such as Chinese Hamster Ovary (CHO) cell line and human peripheral lymphocytes after exposure to γ-radiation. The measurements on the induction of γ-H2AX foci and micronuclei frequency in the cell nuclei indicated that pretreatment with DSePA significantly prevented the radiation induced DNA damage or genotoxicity and subsequent cytotoxicity without exerting its own toxicity. The maximum protective effect of DSePA was seen at a pre-treatment concentration of 3μg/ml. The mechanistic investigations in CHO cells revealed that DSePA pretreatment prevented the radiation induced ROS generation, lipid peroxidation and subsequent apoptosis in these cells. Further, it was seen to augment the mRNA expressions of GPx2 significantly and GPx4 marginally without causing much change in the total GPx activity after radiation exposure. These results suggested the roles of GPx2 and GPx4 in DSePA mediated radioprotection. In conclusion our results confirm the nongenotoxic nature of the DSePA and validate its radioprotective efficacy and mechanisms of action in model cellular systems.