It is well documented that exitotoxicity induced by N-methyl-D-aspartate (NMDA) receptor activation plays a pivotal role in delayed neuronal death in the hippocampal CA1 region after transient global ischemia. However, the effect of gamma-aminobutyric acid (GABA) receptor activation is uncertain in ischemia brain injury. The aim of this study was to investigate whether the enhancement of GABA receptor activity could inhibit NMDA receptor-mediated nitric oxide (NO) production by neuronal NO synthase (nNOS) in brain ischemic injury. The results showed that both the GABA(A) receptor agonist muscimol and the GABA(B) receptor agonist baclofen had neuroprotective effect, and the combination of two agonists could significantly protect neurons against death induced by ischemia/reperfusion. Coapplication of muscimol with baclofen not only enhanced nNOS (Ser847) phosphorylation but also increased the interaction of nNOS with PSD95 at 6 hr and 1 day of reperfusion. Interestingly, the inhibitors of calcineurin and PP1/PP2A could enhance nNOS phosphorylation at Ser847 site at 1 day of reperfusion after ischemia but not at 6 hr of reperfusion. From these data, we conclude that GABA receptor activation could exert its neuroprotective effect through increasing nNOS (Ser847) phosphorylation by different mechanisms at 6 hr and 1 day of reperfusion. The increased interaction of nNOS and postsynaptic density-95 induced by GABA agonists is responsible for nNOS (Ser847) phosphorylation at both time points, but at 1 day of reperfusion the inhibition of protein phosphatase activity by GABA agonists also contributes to the neuroprotection. Our results suggest that GABA receptor agonists may serve as a potential and important neuroprotectant in therapy for ischemic stroke.