Stem- and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. Several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (EVs). EVs are small, cell-derived lipid bilayer enclosed particles that play a role in intercellular communication through their enrichment in specific components like nucleic acids and proteins. Indeed, cardiac progenitor cell (CPC)-derived EVs have been shown to protect the myocardium against ischemia/reperfusion injury. However, the underlying mechanisms for CPC-EV-mediated cardioprotection remain elusive. Here, we utilized the proteomic composition of CPC-EVs released during different culture conditions, to unravel protein-mediated effects of CPC-EVs on the endothelium. CPCs were stimulated with calcium ionophore (ca ion-EVs), previously shown to influence EV release, or vehicle (control-EVs) for 24 hours and EVs were isolated using size exclusion chromatography (SEC). EV concentration and size was assessed using NTA and proteomic composition was profiled using mass spectrometry in triplicate. EVs isolated from SKOV-3 cells were included as a non-stem cell control. Proteomic analysis identified multiple proteins uniquely expressed or enriched in control-EVs compared with ca ion-EVs and SKOV-3 EVs. To better understand the effect that these EVs have on cardioprotection, endothelial cells HMEC-1 were stimulated with CPC-EVs, CaIon-EVs or PBS for 30 min. Phosphorylation of ERK1/2 and AKT, and wound closure was increased in HMEC-1 after stimulation with CPC-EVs but not with Ca ion-EVs. To gain further detail in the pathway activation in HMEC-1 cells, we also performed (phospho)proteomic analysis of these cells upon EV-stimulation. The proteomic and phosphoproteomic changes derived from EV-stimulation in HMEC-1 were quantified by mass spectrometry and validated the activation of PI3K-AKT-mTOR and (Insulin/IGF-) MAPK signaling pathways.