Objective To investigate the roles of endothelial nitric oxide synthase（eNOS） and phosphatidylinositol-3 kinase/protein kinase B（PI3K/Akt） pathway in the neuroprotection of remote ischemic postconditioning (RIPoC) on global cerebral ischemia/reperfusion (I/R) injury in rats. Methods One hundred male adult SD rats weighing 200 g -250 g were randomly divided into 5 groups (n=20 each): group sham, group I/R, group I/R+RIPoC, group L-NAME+I/R+RIPoC, group LY+I/R+RIPoC. Global cerebral I/R was induced by four-vessel occlusion. Group I/R+RIPoC, group L-NAME+I/R+RIPoC and group LY+I/R+RIPoC received 3 cycles of 15 min ischemia in bilateral femoral arteries at the beginning of cerebral reperfusion followed by 15 min reperfusion. The group L-NAME+I/R+RIPoC:4-VO with coinjection of N-nitro-L-arginine methyl eater(L-NAME), 5 mg/kg, in normal saline, intraperitoneslly, 10 min before 4-VO, then followed by RIPOC and 48 h and 7 d of reperfusioin. The group LY+I/R+RIPoC:I/R+RIPoC with injection of LY294002(a highly selective inhibitor of PI3K, LY, 10 uL, 10 mol/L,in 3% DMSO,intracerebroventricularly, 10 min before 4-VO), then followedby RIPOC and 48 h and 7 d of reperfusion. The rats were sacrificed at 48 h of cerebral reperfusion, and brains were removed for determination of neuronal apoptosis [by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL） method] in hippocampal CA1 region and p-eNOS, eNOS, p-Akt and Akt expression (by Western blot) in hippocampal CA1 region. Morris water maze task was used to test the learning and memory function at 4 d of cerebral reperfusion, and the rats were sacrificed at 7 d of cerebral reperfusion, and brains were removed for determination of neuronal density in hippocampal CA1 region. Results Compared with group sham, the number of apoptotic neurons in CA1 region [(0.8±0.8), (84.7±6.8), (52.8±7.8), (74.3±9.0), (79.5±7.3)/mm] increased (P<0.01), learning and memory function decreased (P<0.01) and neuronal density in CA1 region[ (193±7), (10±7), (91±11), (38±7), (26±7)/mm]decreased (P<0.01) in group I/R, I/R+RIPoC, L-NAME+I/R+RIPoC and LY+I/R+RIPoC. RIPoC significantly attenuated these cerebral I/R-induced changes in apoptotic neurons (P<0.01), learning and memory function (P<0.01) and neuronal density (P<0.01). Pre-administration of N(ω)-nitro-l-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) significantly abolished the neuroprotective effect of RIPoC, and significantly attenuated the RIPoC induced up-regulation of p-eNOS (0.48±0.03, 0.23±0.04) and eNOS (0.91±0.07, 0.64±0.06) in CA1 region (P<0.01). Moreover, pre-administration of LY not only significantly reversed the RIPoC induced neuroprotective effect and up-regulation of p-Akt（0.74±0.06, 0.44±0.04）in CA1 region, but also obviously inhibited the RIPoC induced up-regulation of both p-eNOS (0.48±0.03, 0.24±0.04) and eNOS (0.91±0.07, 0.63±0.06) in CA1 region (P<0.01). Conclusions RIPoC protects the brain against global cerebral I/R injury and that this neuroprotection is mediated by up-regulation and activation of eNOS through the PI3K/Akt pathway.