MSC transplantation after I/R injury reduces infarct size and improves cardiac function. There is evidence that this cardioprotective effect is mediated by paracrine signalling, but the mechanism remains yet to be determined. Isolated mouse ventricular myocytes (VMs) were used in an in vitro model of I/R to determine the effect of MSC conditioned tyrode (ConT) on the recovery of VMs from an ischemic challenge (15 min). Measurements of the mitochondrial membrane potential (Ψmito) with TMRM (100 nM), the intracellular Ca concentration (fluo-4/AM) and cell shortening were used as functional readouts. During ischemia VMs exhibit a depolarization of Ψmito and an increase in diastolic calcium concomitant to a decrease in cell contractility. Reperfusion with either Ctrl or ConT resulted in an increase in Ca transient amplitudes. Early After Depolarizations (EADs) frequently observed in Ctrl cells were reduced during ConT reperfusion (EADs: ConT:1% vs. Ctrl: 24%; at 1 min). ConT prolonged VM survival (ConT: 58% vs Ctrl: 33%; at 20 min) and Ca transients returned to pre-ischemic values. After I/R, Ψmito rapidly recovered in Ctrl as well as ConT; however, in Ctrl an exaggerated hyperpolarization of Ψmito (Ctrl: 6 of 9; ConT: 0 of 5 cells) was observed. This hyperpolarization could be prevented by supplementing Ctrl solution after I/R with the ROS scavenger mitoTEMPO (5 μM) or the IK,ATP opener (diazoxide, 200 μM). Enhanced hyperpolarization was induced by supplementing ConT with a blocker of IK,ATP 5-HD (500 μM) or PI3K/Akt inhibitors (LY: 10 μM; Akt iV: 20 μM). In conclusion we could demonstrate that MSC ConT protects VMs from I/R injury by attenuation of arrhythmic Ca release events and by delaying the recovery of Ψmito through PI3K/Akt mediated opening of IK,ATP.