Introduction: Mesenchymal Stromal/Stem Cells (MSC) have been widely used for their therapeutic properties in acute myocardial infarction (AMI) due to their pleiotropic anti-inflammatory, anti-apoptotic, anti-fibrotic and pro-angiogenic properties. Despite promising results in animal studies and their safety/efficacy in phase I/II trials, inconsistencies have been reported in phase III. We have recently shown in a mouse model of ischemia-reperfusion (IR) that the cardioprotective effect of MSC administered during reperfusion was dependent on the presence of PPARβ/δ receptors. However, the role of the modulation of PPARβ/δ on MSC anti-apoptotic and cardioprotective properties has never been investigated. Objectives: this study aims investigating the role of PPARβ/δ activation on MSC therapeutic properties in an ex vivo model of AMI and their anti-apoptotic functions on cardiac cells.Method: isolated mouse hearts were subjected to 30 minutes of global ischemia and 60 minutes of reperfusion (IR). Murine MSC (naïve MSC), MSCago (pretreated with GW0742 1 µM agonist) MSCantago (pretreated with GSK0660 1µM) were administered in the perfusion system during the reperfusion period. Specific DNA fragmentation was quantified in MSC or MSCago challenged with 350 µM H2O2 during 4 hours and in H9c2 rat cardiomyoblasts and EAhy926 human endothelial cells challenged with H2O2 and co-cultured with naïve or primed MSC.Results: MSCago injection during reperfusion decreased infarct size by a larger extent than MSC, an effect lost with MSCantago. Accordingly, an increased number of cells of MSCago was detected in the left ventricular wall after 1 hour of reperfusion. In vitro, we observed for MSCago an increased resistance against oxidative resistance and a more potent anti-apoptotic effect on both cardiomyocytes and endothelial cells in vitro than naive MSC.Conclusion: MSC preconditioning via PPARβ/δ enhanced their cardioprotective effect against IR injury ex vivo by both increasing their resistance to oxidative stress and their anti-apoptotic effects on myocytes and endothelial cells. These results could be of major interest to improve MSC efficacy for the cardioprotection of the myocardium in AMI patients.