Abstract: AIM To explore the role of phosphatidylinositol 3′-kinase (PI3-K)/protein kinase B (Akt) signaling pathway during DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) inhibited cardiomyocytes apoptosis induced by ischemia/reperfusion injury (I/RI). METHODS Cardiomyocytes apoptosis was induced by I/RI, then it was treated with phosphatidylinositol 3 kinas’ inhibitor LY294002 ［22(42-Morpholine) 282 H-24-212 benzopyran 242］(specific inhibitor of PI3-K). Experiment was divided into the normal control group, I/RI group, I/RI+DIDS group and I/RI+DIDS+LY294002 group. The cell viability, morphology changes of nucleus and caspase-3 activity, Akt phosphorylation was observed through the MTT colorimetric, Hoechest-33258 staining and caspase (Apo-ONETM Homogeneous Caspase)-3 kit and Western blot. RESULTS ①DIDS was able to markedly inhibit the I/RI indicated by a decline of cell viability, inhibited the emergence of apoptotic bodies, restrained the increase of caspase-3 activity (P<0.01); ②Pretreatment with LY294002, the increase of myocardial cells’ viability, decrease of apoptotic bodies and the increase of caspase-3 activity were significant blocked (P<0.01); ③There was no significant difference of Akt phosphorylation between I/RI group and control. DIDS could dramatically increase Akt phosphorylation in I/RI group (P<0.01); Pretreatment with LY-294002, DIDS’ effect on the Akt phosphorylation in the I/RI group was decreased significantly (P<0.01). CONCLUSION DIDS attenuates I/RI-induced myocardial apoptosis through the activation of PI3K/Akt signaling pathway.