Radiotherapy is one of the most common therapies for treating human cancers. Several studies have indicated that irradiation induces reactive oxygen species (ROS), which play an important role in radiation damage of the cell. It has been shown that Nigella sativa L. (NS) and reduced glutathione (GSH) have both an antiperoxidative effect on different tissues and a scavenger effect on ROS. The purpose of this study was to determine the antioxidant and radioprotective roles of NS and GSH against irradiation-induced oxidative injury in an experimental model. The NS group was administrated NS (1 mL/kg body weight), the GSH group was injected GSH (150 mg/kg body weight) and the control group was given physiologic saline solution (1 mL/kg body weight) for 30 consecutive days before exposure to a single dose of 6 Gy of radiation. Animals were sacrificed after irradiation. Malondialdehyde, nitrate, nitrite (oxidative stress markers) and ascorbic acid, retinol, β-carotene, GSH and ceruloplasmin (nonenzymatic antioxidant markers) levels and peripheral blood lymphocytes were measured in all groups. There were statistically significant differences between the groups for all parameters (P < 0.05). Whole-body irradiation caused a significant increase in blood malondialdehyde, nitrate and nitrite levels. The blood oxidative stress marker levels in irradiated rats that were pretreated with NS and GSH were significantly decreased; however, nonenzymatic antioxidant levels were significantly increased. Also, our results suggest that NS and GSH administration prior to irradiation prevent the number of alpha-naphthyl acetate esterase peripheral blood T lymphocytes from declining. These results clearly show that NS and GSH treatment significantly antagonize the effects of radiation. Therefore, NS and GSH may be a beneficial agent in protection against ionizing radiation-related tissue injury.