Ischemic stroke interrupts the flow of blood to the brain and subsequently results in cerebral infarction and neuronal cell death, leading to severe pathophysiology. Glutathione (GSH) is an antioxidant with cellular protective functions, including reactive oxygen species (ROS) scavenging in the brain. In addition, GSH is involved in various cellular survival pathways in response to oxidative stress. In the present study, we examined whether GSH reduces cerebral infarct size after middle cerebral artery occlusion in vivo and the signaling mechanisms involved in the promotion of cell survival after GSH treatment under ischemia/reperfusion conditions in vitro. To determine whether GSH reduces the extent of cerebral infarction, cell death after ischemia, and reperfusion injury, we measured infarct size in ischemic brain tissue and the expression of claudin-5 associated with brain infarct formation. We also examined activation of the PI3K/Akt pathway, inactivation of FOXO3, and expression of Bcl2 to assess the role of GSH in promoting cell survival in response to ischemic injury. Based on our results, we suggest that GSH might improve the pathogenesis of ischemic stroke by attenuating cerebral infarction and cell death.