The capacity of the luminal layer of an electrospun bi-layer scaffold composed of gelatin, elastin, polycaprolactone (PCL), and poliglecaprone (PGC) to promote endothelial regeneration was investigated using human aortic endothelial cells (HAECs). HAECs of different densities were cultured on a thin film of the luminal layer of the scaffold mounted on a cell crown for desired periods. Fluorescent images showed that HAECs formed a mono-layer within 24 h after having successfully adhered to the scaffold's surface. Scanning electron microscopy (SEM) revealed a satisfactory coverage by the HAECs. Death rates of HAECs populations determined by fluorescent staining were below 5% within the initial 3 days while the profile of proliferation exhibited an exponential increase within 11 days as determined by the 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide (MTT) assay. The functionalities of the endothelial mono-layer were probed by ZO-1 staining for tight junction formation, by 6-keto-PGF1α assay for prostacyclin (PGI2) secretion, and by human platelets for its anti-thrombotic capability. The results indicated that the regenerated endothelium possessed normal functions associated with native endothelium. This study suggests that this electrospun bi-layer scaffold is a promising candidate for cardiovascular grafting for its capability of promoting the regeneration of a functional endothelium to prevent blood clotting in small diameter grafts. © 2010 Elsevier Ltd.