3,3'-Diselenodipropionic acid (DSePA), a diselenide and a derivative of selenocystine. was evaluated for in vivo radioprotective effects in Swiss albino mice, at an intraperitoneal dose of 2 mg/kg body wt, for 5 days before whole-body exposure to g-radiation. The radioprotective efficacy was evaluated by assessing protection of the hepatic tissue, the spleen, and the gastrointestinal (GI) tract and survival against sub- and supralethal doses of g-radiation. DSePA inhibited radiation-induced hepatic lipid peroxidation, protein carbonylation, loss of hepatic function, and damage to the hepatic architecture. DSePA also attenuated the depletion of endogenous antioxidants such as glutathione, glutathione peroxidase, superoxide dismutase, and catalase in the livers of irradiated mice. DSePA also restored the radiation-induced reduction in villus height, crypt cell numbers, and spleen cellularity, indicating protective effects on the GI tract and the hematopoietic system. The results from single-cell gel electrophoresis of the peripheral blood leukocytes showed that DSePA can attenuate radiation-induced DNA damage. The mRNA expression analysis of genes revealed that DSePA augmented GADD45a and inhibited p21 in both spleen and liver tissues. DSePA also inhibited radiation-induced apoptosis in the spleen and reversed radiation-induced alterations in the expression of the proapoptotic BAX and the antiapoptotic Bcl-2 genes. In line with these observations, DSePA improved the 30-day survival of irradiated mice by 35.3%. In conclusion, these findings clearly confirm that DSePA exhibits protective effects against whole-body g-radiation and the probable mechanisms of action involve the maintenance of antioxidant enzymes, prophylactic action through the attenuation of the DNA damage, and inhibition of apoptosis.