Background and Purpose— Downregulation of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is thought to be a novel neuroprotective strategy in ischemic stroke, but the underlying mechanisms remain unclear. In this study, we aimed to validate the use of PTEN regulation of γ-aminobutyric acid subtype A receptors (GABA _A Rs) as a molecular target for the treatment of ischemic stroke. Because suppression of GABA _A Rs contributes to ischemic neuron death, describing the intracellular signaling that interacts with GABA _A Rs in ischemic neurons would provide a molecular basis for novel stroke therapies. Methods— We measured surface GABA _A R expression by immunocytochemical labeling and surface protein biotinylation assay. Knockdown and overexpression approaches were used to test the effects of PTEN on the expression and function of GABA _A Rs. Neuronal death was detected in both in vitro and in vivo stroke models. Results— The knockdown and overexpression approaches provided the first evidence that PTEN negatively regulated membrane expression and function of GABA _A Rs in rat hippocampal neurons. Importantly, we demonstrated that a PTEN inhibitor prevented the reduction of surface GABA _A Rs in injured hippocampal neurons subjected to oxygen-glucose deprivation, an in vitro insult that mimics ischemic injury, whereas a GABA _A R antagonist significantly reduced this PTEN inhibitor–induced neuroprotection in both the in vitro and in vivo ischemic stroke models. Conclusions— Our study provides direct evidence that downregulation of PTEN protects against ischemic neuron death by preserving GABA _A R function. Targeting this pathway may be an effective strategy for development of selective, potent stroke treatments.