Increasing evidence demonstrates that oxidative stress plays an important role in brain injury in experimental models of brain ischemia. Thymoquinone, the main constituents of the volatile oil from Negella sativa seeds, is reported to possess strong antioxidant properties. Hence, the present study was undertaken to evaluate the neuroprotective effect of thymoquinone against transient forebrain ischemia-induced neuronal damage in the rat hippocampus. Rats were divided randomly into five groups: control, sham, ischemia, thymoquinone and ischemia + thymoquinone. Transient forebrain ischemia was induced with bilateral occlusion of both common carotid arteries for 10 min followed by 7 days of reperfusion. Thymoquinone was administered (5 mg/kg/day p.o.) 5 days before ischemia and continued during the reperfusion time. Animals were sacrificed, and brain tissues were isolated for histopathological examination. Hippocampal tissues were also used for determination of malondialdehyde levels, an end product of lipid peroxidation; glutathione (GSH) levels, a key antioxidant and the activities of the antioxidant enzymes catalase and superoxide dismutase (SOD). Thymoquinone and its metabolite thymohydroquinone were tested as inhibitors of the in vitro non-enzymatic lipid peroxidation induced by iron-ascorbate in the hippocampal homogenate. Forebrain ischemia–reperfusion neural injury in rats was demonstrated by histopathological observation, which revealed significant neural cell death in the hippocampus CA1 area 7 days post-ischemia (77% cell loss). Additionally, forebrain ischemia–reperfusion oxidative injury in rats was demonstrated by a significant increase in malondialdehyde and a significant decrease in GSH contents, catalase and SOD activities in the hippocampal tissue compared to the control or sham-operated groups. Pretreatment of thymoquinone attenuated forebrain ischemia-induced neuronal damage manifested by significantly decreasing the number of dead hippocampal neuronal cells (24% in thymoquinone-treated versus 77% for ischemia, P < 0.001), which confirm the protective role of thymoquinone in ischemia–reperfusion injury. Also, pretreatment of ischemic rats with thymoquinone decreased the elevated levels of malondialdehyde and increased GSH contents, catalase and SOD activities to normal levels. Thymoquinone and thymohydroquinone inhibited the in vitro non-enzymatic lipid peroxidation in hippocampal homogenate induced by iron-ascorbate. The IC50 for thymoquinone and thymohydroquinone were found to be 12 and 3 μM respectively. This suggests that the protection of thymoquinone and its metabolite involve increased resistance to oxidative stress. In conclusion, thymoquinone is effective in protecting rats against transient forebrain ischemia-induced damage in the rat hippocampus. This spectacular protection makes thymoquinone a promising agent in pathologies implicating neurodegenaration such as cerebral ischemia.