Exercise is an efficient strategy for myocardial protection against ischemia–reperfusion (IR) injury. Although endothelial nitric oxide synthase (eNOS) is phosphorylated and activated during exercise, its role in exercise-induced cardioprotection remains unknown. This study investigated whether modulation of eNOS activation during IR could participate in the exercise-induced cardioprotection against IR injury. Hearts isolated from sedentary or exercised rats (5 weeks training) were perfused with a Langendorff apparatus and IR performed in the presence or absence of NOS inhibitors [N-nitro-l-arginine methyl ester, l-NAME or N5-(1-iminoethyl)-l-ornithine, l-NIO] or tetrahydrobiopterin (BH₄). Exercise training protected hearts against IR injury and this effect was abolished by l-NAME or by l-NIO treatment, indicating that exercise-induced cardioprotection is eNOS dependent. However, a strong reduction of eNOS phosphorylation at Ser1177 (eNOS-P^Ser1177) and of eNOS coupling during early reperfusion was observed in hearts from exercised rats (which showed higher eNOS-P^Ser1177 and eNOS dimerization at baseline) in comparison to sedentary rats. Despite eNOS uncoupling, exercised hearts had more S-nitrosylated proteins after early reperfusion and also less nitro-oxidative stress, indexed by lower malondialdehyde content and protein nitrotyrosination compared to sedentary hearts. Moreover, in exercised hearts, stabilization of eNOS dimers by BH₄ treatment increased nitro-oxidative stress and then abolished the exercise-induced cardioprotection, indicating that eNOS uncoupling during IR is required for exercise-induced myocardial cardioprotection. Based on these results, we hypothesize that in the hearts of exercised animals, eNOS uncoupling associated with the improved myocardial antioxidant capacity prevents excessive NO synthesis and limits the reaction between NO and O₂ ^·− to form peroxynitrite (ONOO⁻), which is cytotoxic.