BACKGROUND Previous studies have suggested that implantation of mesenchymal stem cells (MSC) or their conditioned media (MSC CM) improves heart function after myocardial infarction. We sought to determine whether MSC and MSC CM added at the onset of reperfusion attenuates myocardial reperfusion injury. METHODS Rat MSC and neonatal rat cardiomyocytes (NRC) were isolated and cultured separately. NRC were subjected to simulated in vitro ischemia/reperfusion (I/R). At the onset of reperfusion, NRC received either fresh medium (control group) or one of the following treatments: MSC in fresh medium; MSC CM alone (without MSC); MSC CM + inhibitors of PI3K (LY294002 or Wortmannin); MSC CM + antibodies neutralizing IGF-1 or VEGF; MSC + inhibitors of PI3K; or cyclosporine. Cell injury was assessed by LDH activity and MTT staining at the end of reperfusion. VEGF, IGF-1 and HGF were measured in each experimental treatment preparation. Ex vivo experimentation on isolated rat hearts subjected to I/R were performed to evaluate the protective effects of MSC CM on myocardial reperfusion injuries measured through CK release and infarct size after TTC staining. RESULTS In vitro cell injury was significantly reduced by MSC, MSC CM and CsA. PI3K inhibitors significantly attenuated the protection afforded by MSC CM but not growth factor inhibitors. Ex vivo experimentation showed that MSC CM significantly reduced myocardial I/R injury. CONCLUSIONS Our data suggest that MSC CM added at the onset of reperfusion can protect myocardium from I/R injury. In vitro data suggest a protection mediated by paracrine activation of the PI3K pathway.