Objectives: Many studies showed that sildenafil have cardioprotective effects mediated by nitric oxide and ischemic preconditioning. Mitochondria play critical roles in both the life and death of cardiac myocytes. We tested whether sildenafil could make rat hearts resistant to infarction through mitochondrial protection using atomic force microscopy (AFM). Methods: To prove the cardiac protective effect of sildenafil and investigate the morphologic and property analysis of mitochondria by AFM in the rat myocardium, in-vivo myocardial infarction (MI) model were used. Rat hearts were subjected to 40 min local ischemia by ligation of the left anterior descending (LAD) coronary and examined infarct size after 5 days of reperfusion. Isolated mitochondria were dropped onto a mica surface and AFM imaging was performed using the non-contact mode of NANOS N8 NEOS (Bruker, Herzogenrath, Germany). The effect of sildenafil on myocardial protection was assessed by TTC staining, TUNEL and immunoblot analysis with anti- bax, bcl-2 and caspase-3 antibodies. RESULTS: Infarct area was significantly reduced in sildenafil-treated rats (7.78 짹 3.9% vs. 20.37짹 7.0% in sildenafil and control hearts, respectively, P < 0.001) as in the previous studies. Thus a relative reduction of 62% in the infarcted zone was observed in the sildenafil-treate rats. From the shape parameters of mitochondria in AFM image, it seemed that myocardial infarction caused the mitochondrial swelling (1,495 짹 1,139 nm2 in normal vs. 24,150 짹 18,289 nm2 in MI, p < 0.0001). Whereas sildenafil reduced the mitochondrial area (7,428 짹 3,682 nm 2, p < 0.0001) by 69.23% compared to that of MI. In addition, sildenafil-mediated cardioprotection was associated with mitochondrial KATP channel. Conclusions: In MI rat model, cardioprotective effect of sildenafil pretreatment associated with a mitochondrial protective mechanism.