Aim To explore the effect of chloride channel blocker DIDS on cell signaling pathway phosphatidylinositol 3'-kinase/proteinase B (PI3K/Akt) and its downstream molecules endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) in staurosporine (STS)-treated cardiomyocyte apoptosis. Methods Neonatal rat cardiomyocytes were exposed to STS in the presence or Absence of DIDS. Cell viability,apoptosis and expressions of Akt,phospho-Akt (p-Akt),eNOS,phospho-eNOS (p-eNOS) and NO production were determined. Results DIDS markedly improved cell viability on STS-exposed cardiomyocytes. DIDS resulted in a 2.1-fold increase of p-Akt over control levels,prevented the reduction in eNOS expression and phospho-eNOS levels induced by STS and significantly increased NO production (all P<0.01). Pretreatment with LY294002,a selective PI3K inhibitor,abolished DIDS-induced increases in p-Akt,eNOS,p-eNOS and NO production,and completely abrogated the DIDS-induced anti-apoptotic effect (P<0.01). Pretreatment with L-NAME,a non-selective NOS inhibitor similarly inhibited the increased NO but only partly abolished protective effects of DIDS (P<0.05). Conclusion DIDS inhibits STS-induced cardiomyocyte apoptosis via activating PI3K/Akt signaling pathway.