Ionizing radiation-induced free radicals cause functional and structural harmful effects. Thiol, an important antioxidant, plays a major role in the eradication of reactive oxygen molecules. Thiol/disulphide homeostasis is a marker of oxidative stress. The objective of this study was to assess the potential radioprotective effects of thymoquinone (TQ) on the dynamic thiol/disulphide homeostasis of rats receiving total-body irradiation (IR). Twenty-two rats were divided into three groups to test the radioprotective effectiveness of TQ. The sham control group did not receive TQ or IR. The IR group received only total-body IR. The TQ + IR group received IR plus TQ. Following IR, blood samples were taken. The thiol/disulphide homeostasis parameters were analysed by a newly established method. In the IR group, native thiol and the native thiol/total thiol ratio were significantly decreased (P = 0.003 and P = 0.003, respectively), whereas the disulphide/native thiol and disulphide/total thiol ratios were significantly increased when compared with those of the sham control group (P = 0.003 and P = 0.003, respectively). In the TQ + IR group, the mean disulphide, native thiol and total thiol levels and the disulphide/native thiol, disulphide/total thiol and native thiol/total thiol ratios were not found to be significantly different when compared with those of the sham control group (P > 0.05 for all). Thiol/disulphide homeostasis was found to be disturbed after IR exposure. The results showed that TQ had antioxidant effects and reduced the IR-induced oxidative stress, which was demonstrated through the dynamic thiol/disulphide homeostasis. Thus, the use of TQ before radiation treatment helped protect the rats from oxidant side effects.