Purpose: To evaluate and optimize the radioprotective ability of the most potent fraction of an aqueous extract of Coronopus didymus in whole body γ-irradiated Swiss albino mice and to evaluate the antioxidant status and lipid peroxidation of the livers of the surviving mice. To correlate the free radical scavenging studies with in vivo radioprotection ability. Materials and methods: Swiss albino mice were treated with either vehicle or the different doses of extract/fraction suspension by an i.p. route, 30 min before exposure to 10 Gy γ-irradiation and the animals were monitored twice daily for any signs of radiation toxicity and mortality. Radiation dose response (7-11 Gy), optimization of route, time of drug administration and evaluation of dose response factor (DRF) at the best dose of the fraction was studied. Endogenous antioxidant status and lipid peroxidation of the livers of the mice surviving on the 31st day was evaluated by using spectrophotometric methods. Results: The most active free radical scavenging fraction (CDF1) as assessed by competition kinetic studies using pulse radiolysis showed maximum in vivo radioprotection of 70% at a dose of 400 mg/kg body weight (bw) compared to corresponding 10 Gy irradiated control. Optimum radioprotection was observed upon i.p. administration, 30 min prior to 10 Gy irradiation and DRF at a dose of 400 mg/kg bw for 30 day survival was found to be 1.07. The levels of endogenous antioxidant enzymes and lipid peroxidation in the CDF1 treated surviving mice were found to reverse back to their normal levels. Conclusions: The optimum dose, time and route of drug administration for maximum radioprotection by CDF1 were determined. The reversal of the levels of endogenous antioxidant enzymes and lipid peroxidation indicates reduced oxidative stress in CDF1 treated surviving mice. © 2006 Informa UK Ltd.