Coronary artery disease (CAD) remains a leading cause of death worldwide. Myocardial ischemia-reperfusion (IR) injury is a major pathological event leading to morbidity and mortality in those with CAD. It is well established that repeated bouts of exercise improve myocardial tolerance to IR, termed exercise-induced cardioprotection. However, gaps remain in our understanding of this cardioprotection, including: sex differences, dose-response, and the cellular mechanisms involved. Therefore, we examined the cardioprotective effects of short- and long-term exercise against myocardial ischemia-reperfusion injury in male and female rats, while investigating unexplored cellular mechanisms. Male and female rats were subjected to 0 days (Sed), 5 days (EX5d), or 8 weeks (EX8w) of treadmill running. Maximal capacity exercise tests were performed before and after the training protocols. EX5d animals did not improve exercise capacity, while EX8w animals did, with no difference between the sexes in % improvement. Following exercise training, rats underwent regional myocardial ischemia-reperfusion (45min/24hr), and infarct size was measured for each heart. Sed females had smaller infarcts than Sed males. Short-term running reduced infarct size in males and females compared to Sed. Long-term running did not reduce infarct size in males compared to EX5d, but EX8w females exhibited smaller infarcts compared to Sed and EX5d females. Myocardial calpain and matrix metalloproteinase-2 (MMP-2) protein levels were not different between experimental groups, but calpastatin and Tissue inhibitor of metalloproteinase-2 (TIMP-2) levels were increased in EX groups compared to Sed, with no difference between sexes. Females had higher protein levels of both subunits of the KATP channel (Kir6.2, SUR2A) compared to males across all experimental groups. We also identified expression level changes of several microRNAs in response to IR and modifications to these changes associated with exercise. The major findings of these studies are: (1) changes in exercise capacity are not required for exercise-induced cardioprotection against IR injury; (2) long-term exercise augmented cardioprotection against IR injury compared to short-term exercise, but only in females; (3) exercise increased levels of TIMP-2; (4) exercise altered expression levels of microRNAs involved with IR injury; and (5) expression of MMP-2 was upregulated in the ischemic region compared to the unaffected area.