The epicardium is a single layer of epithelial (mesothelial) cells that covers the entire heart surface, but whose function in adult mammals is poorly understood. Defining the role of epicardial cells during homeostasis, growth and injury has potential to provide new treatment strategies for human diseases that result in heart failure, due to extensive loss of viable cardiac tissue. We hypothesized that epicardial cells contribute to repair as transplantable progenitor cells for cellular regeneration and as a source of secreted growth factors for cell protection after myocardial infarction. Adult epicardial cells were prospectively isolated as uncommitted epithelial cells using epithelial-specific beta-4 integrin (CD104). These cells underwent epithelial to mesenchymal transformation in culture to generate epicardial cell derivatives (EPDCs). We demonstrate that the C-terminal peptide from Connective Tissue Growth Factor (CTGF-D4), when combined with insulin, effectively primes EPDCs for robust cardiac engraftment in rats and contributes to improvement in cardiac function at one month after MI. Furthermore, we define a signaling axis comprised of CTGF-D4, low density lipoprotein receptor-related protein 6 (LRP6), sex determining region Y-box 9 (Sox9) and Endothelin Receptor B (ETBR) that controls several key processes that impact EPDC graft success: cell survival, proliferation and migration. Interestingly, conditional deletion of ETBR using epicardial-specific transgenic mice prevented epicardial cell proliferation and migration into myocardium after MI. We therefore observed a congruence in the signals and signaling pathways that control the proliferation and migration of endogenous EPDCs after MI and EPDCs that can be generated in cell culture and grafted back to the heart. To gain additional insight into the cellular contribution of the epicardium, we utilized a non-injurious running exercise model to evaluate epicardial activity as a consequence of cardiac hypertrophy (i.e. myocardial growth model). We employed an inducible lineage-tracing system to specifically label and track epicardial cells by GFP expression. Prolonged exercise resulted in a significant number of GFP-positive proliferating epicardial cells and epicardial-derived GFP-positive endothelial cells and few GFP-positive smooth muscle cells in the heart. These observations highlight the cellular plasticity of the adult epicardium and its function as a cardiac progenitor cell niche, maintaining a source of replacement cells. To investigate the paracrine properties of adult epicardial cells for their role in cell protection after MI and reperfusion, human epicardial cells were isolated from donor atrial tissue explants. We predicted that medium conditioned by cultured epicardial cells (EPI CdM) contained secreted reparative factors that would promote endothelial cell survival. Administration of EPI CdM promoted endothelial cell survival in culture and in vivo, 24 hours after ischemia-reperfusion injury. By screening EPI CdM, we detected protein complexes containing hepatocyte growth factor (HGF) with polyclonal IgG that imparted vascular protection in vivo in a manner similar to EPI CdM. Overall, the studies presented here illustrate the unique biology of epicardial cells, their signaling networks, and their contribution to cardiac cell protection and regeneration. Importantly, these properties have the potential to be exploited in translational applications for cardiac repair.