We investigated the mechanism of exercise-induced late cardioprotection against ischemia/reperfusion (I/R) injury. C57BL/6 mice received treadmill exercise (60 min/day) for 7 days at a work rate of 60-70% maximal O₂ uptake. Exercise transiently increased oxidative stress and activated endothelial isoform of nitric oxide synthase (eNOS) during exercise and increased expression of inducible isoform of NOS (iNOS) in the heart after 7 days of exercise. The mice were subjected to regional ischemia by 30 minutes occlusion of left coronary artery followed by 2 hours reperfusion. Infarct size was significantly smaller in the exercised mice. Ablation of cardiac sympathetic nerve (CSN) by topical application of phenol abolished oxidative stress, activation of eNOS, upregulation of iNOS, and cardioprotection mediated by exercise. Treatment with the antioxidant N-(2-mercaptopropionyl)-glycine (MPG) during exercise also inhibited activation of eNOS, upregulation of iNOS and cardioprotection. In eNOS^-/- mice, exercise-induced oxidative stress was conserved but upregulation of iNOS and cardioprotection were lost. Exercise did not confer cardioprotection when the iNOS selective inhibitor 1400W was administered just before coronary artery occlusion or iNOS^-/- mice were employed. These results suggest that exercise stimulates CSN that provokes redox-sensitive activation of eNOS, leading to upregulation of iNOS which acts as a mediator of late cardioprotection against I/R injury.