The aim of this study was to investigate the effects of hyperbaric oxygenation (HBO) after brain ischemia. Middle cerebral artery occlusion (MCAO) procedure was used to induce the brain ischemia. Rats were assigned to control or HBO group after brain ischemia. In order to examine the role of glutathione after HBO treatment, another group of brain ischemic rats were included to receive the glutathione synthesis inhibitor and HBO treatment. HBO was administered at a pressure of 3 atmospheres absolute for 1 h with 100% oxygen, starting at 3 h post brain ischemia in HBO groups. Animals in control group were placed in their home cage and exposed to normobaric room air. The infarct volume (IV), activation of astrocyte, and level of total glutathione and lipid peroxidation (LP) were assessed 24 h post-reperfusion. Significant reduction in IV was noted in HBO group when compared with control group. The activation of astrocyte was significantly increased in the right cerebral cortex and right striatum in the HBO group when compared with those of the control group. The glutathione level was higher with lower LP level in right cortex and right striatum after HBO as compared with those of the control. However, such effects of HBO treatment were markedly reduced by glutathione synthesis inhibitor administration. These results show that inhibiting glutathione synthesis dramatically reduces the effectiveness of HBO in acute transient focal cerebral ischemia.