【Abstract】 Objective To study the role of PI3K/Akt/eNOS signaling pathway in diazoxide-postconditioning against in vivo rat myocardial ischemia reperfusion injury. Methods 60 SD rats were randomly divided into five groups: sham operation group (S), I/R, diazoxide group (D), inhibitor of PI3K wortmannin group (W) and diazoxide + wortmannin group (D + W). In vivo myocardial I/R injury model was made by ligation of left anterior descending coronary artery 30 min followed by 120 min reperfusion except for S group. Each group was infused respectively with 0.1% DMSO, 0.1% DMSO, diazoxide 7 mg﹒kg-1, wortmannin 15 μg﹒kg-1 and diazoxide via the femoral vein 5 min before reperfusion, and wortmannin was given 5 min before administration of diazoxide in D + W group; all drugs were continuously infused for 15 min. At the end of reperfusion, the plasma concentration of cardiac troponin I (cTnI) was measured; myocardial pathological changes were examined by HE staining; the expression of eNOS was evaluated by immunohistochemical analysis. Results Compared with S group, concentration of cTnI was significantly decreased (P<0.01), myocardium damaged obviously, and the expression of eNOS was markedly increased in other four groups. Compared with I/R group, concentration of cTnI was significantly decreased(P<0.01), myocardial pathological changes were markedly slighter, accompanying with significant high expression of eNOS (P<0.01) in Group D and D + W. Compared with group D, concentration of cTnI was significantly increased (P<0.05), and myocardium damaged more severely with significantly decreased expression of eNOS (P<0.01) in D + W group. Conclusion Diazoxide-postconditioning is capable to reduce myocardial I/R injury induced, which is partially related with the activation of the PI3K/Akt/eNOS signaling pathway.