Progenitor cell plasticity enhances positive remodeling of damaged tissue. We and others have previously shown that progenitor cells may limit apoptosis and modulate inflammation in part by the production of growth factors. However, recent studies suggest that progenitor cells senesce and lose their differentiation potential with increasing time in culture and passage. We hypothesize that murine bone marrow mesenchymal stem cells (MSCs) are cardioprotective against ischemia/reperfusion injury in the isolated perfused rat heart, and that passage number has an adverse effect on MSC activation and cardioprotection. Adult male and female Sprague-Dawley rat hearts were isolated, perfused via Langendorff model, and subjected to ischemia/reperfusion. Mouse MSCs were harvested, cultured, suspended in perfusate, and infused before global index ischemia. Hearts were assigned to controls or infusion with passage 3, 5, or 10 MSCs. In addition, MSCs in culture were stressed by hypoxia and increasing doses of endotoxin (lipopolysaccharide). Mesenchymal stem cell activation was determined by measuring vascular endothelial growth factor production with enzyme-linked immunosorbent assay. All data are reported as mean ± SEM and were analyzed with 2-way analysis of variance. Differences are considered significant if P < 0.05. Passage 3 murine MSC infusion in hearts before ischemia reduced the depression of left ventricular developed pressure, attenuated the increase of end-diastolic pressure, and reduced the depression of +dP/dT and −dP/dT. However, the MSC protective effect disappeared in hearts infused with passage 5 and passage 10 MSCs. Although hypoxia and lipopolysaccharide resulted in significant activation of MSCs, passage 3 MSCs demonstrated significantly greater vascular endothelial growth factor release than passage 5 and 10 MSCs. Acute murine MSC infusion confers protection in isolated rat hearts. However, high passage number has an adverse effect on MSC activation and protection. This portends limited ex vivo expansion before possible therapeutic use.