Thymoquinone (TQ) has been reported as a potent inducer of apoptosis in cancer cells. However, the role of TQ as an apoptotic or antiapoptotic has not been established yet in other types of cell injuries. Our objective was to explore whether TQ exerts a hepatoprotective effect against hepatic ischemia reperfusion injury (I/R) and to identify its potential effect on apoptotic pathways. Rats were divided into eight groups: group I: shamoperated; group II: I/R (45 min ischemia-60 min reperfusion). The other six groups were given PO administration of TQ aqueous solution at 5, 20, and 50 mg/kg/day dose levels for 10 days. At the end of treatment three groups were not subjected to any intervention (groups III, IV, and V: TQ control groups) or subjected to 45 min ischemia followed by one-hour reperfusion as in group II (groups VI, VII, and VIII: TQ pretreated I/R groups). Serum levels of liver enzymes, tissue levels of malondialdehyde (MDA), reduced glutathione (GSH) and TNF-α were measured. Activities of caspases 8, 9, and 3 were determined. Cytochrome c in cytosol was determined by solid phase ELISA. Expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins as well as nuclear factor κB (NF-κB) were assessed using polymerase chain reaction. Apoptosis end point was detected using electrophoresis for analysis of DNA fragmentation. TQ administration before I/R resulted in a significant decrease in liver enzymes, MDA and TNF-α tissue levels with increased GSH content. It also inhibited cytochrome c release into the cytosol, down regulated the expression of NF-κB and Bax and up regulated the Bcl-2 proteins. Hepatic apoptosis was significantly attenuated as indicated by a significant decrease in all caspase activities and by DNA fragmentation. In conclusion, TQ exerts an antiapoptotic effect through attenuating oxidative stress and inhibiting TNF-α induced NF-κB activation. Furthermore, it regulates the Bcl-2/Bax ratio and inhibits downstream caspases in this I/R model.