Abstract Obesity is a major health issue that impedes the ability of preconditioning and postconditioning to protect the myocardium against infarction secondary to dysregulation of kinase signaling pathways. Moreover, exercise decreases cardiovascular mortality in obese patients but the mechanism remains to be established. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise (1 h/day, 5 days/7, 4 weeks, 4° slope, 10–30 cm/s) and underwent 30 min of coronary artery occlusion followed by 24 h of reperfusion for infarct size measurement. In WT, exercise reduced infarct size by 60% and increased phosphorylation of kinases such as Akt, ERK 1/2, p70S6K, AMPK and GSK3?. Importantly, the level of corresponding phosphatases PTEN, MKP-3 and PP2C was decreased. Calcium concentration inducing the opening of mitochondrial permeability transition pore (mPTP) was increased by exercise. In ob/ob, regular exercise induced a robust cardioprotection by reducing infarct size (? 67%), increasing kinase phosphorylation, decreasing phosphatase levels and improving the resistance to mPTP opening. However exercise did not modify hyperglycemia, hypercholesterolemia, hyperinsulinemia, fat mass and body weight in obese mice. In conclusion, regular exercise induces cardioprotection against myocardial infarction despite obesity and restores pro-survival signaling pathways with simultaneous increase in kinase phosphorylations, decreased levels of phosphatases and increased resistance of mPTP opening, independently from improvement in associated co-morbidities.