To date, it is clearly accepted that the activation of endothelial isoform of NOS (eNOS) by exercise training constitutes a key trigger of exercise-induced cardioprotection against ischemia-reperfusion (IR). However, this enzyme is expressed both in coronary endothelial cells and cardiomyocytes, but the contribution of the one or the other one to such cardioprotection has never been challenged. The aim of this study was then to investigate the role of cardiomyocytes vs. endothelial eNOS in exercise cardioprotection. To this, rats were assigned to sedentary (Sed) or chronic aerobic exercised (Ex) group. Effects of exercise on vulnerability to IR or anoxia-reoxygenation (AR) were respectively evaluated at whole heart or cardiomyocytes and coronary artery levels. On a first set of rats, isolated cardiomyocytes were submitted to AR in presence or not of L-NAME, an eNOS inhibitor. Exercise reduced cells death and improved cells contractility after AR, but no effect of L-NAME was observed. Interestingly, exercise had no effect neither on eNOS phosphorylation on its activation site (Ser1177) nor on S-nitrosylation at cardiomyocytes level, whereas at whole heart level exercise increased both of them, suggesting that exercise impacted endothelial cells rather than cardiomyocytes. Then, to evaluate the contribution of endothelial cells on exercise-induced cardioprotection, on a Langendorff apparatus we treated hearts with Triton X-100 before IR to abolish coronary endothelial cells activity. Inactivation of endothelial cells totally suppressed cardioprotective effects of exercise. Finally, coronary arteries were isolated from hearts submitted to IR and endothelial function was assessed on a wire-myograph. We observed that alteration of endothelium-dependent coronary relaxation induced by IR was reduced in Ex group. In conclusion, these results show that coronary endothelial cells rather than cardiomyocytes play a key role in eNOS-dependent cardioprotection in Ex rat hearts.