Endothelial cells (ECs) form a monolayer lining over the entire vascular wall and play an important role in maintaining vascular homeostasis and cancer metastasis. Loss of proper endothelial function can lead to vascular diseases. Therefore, the endothelial monolayer is particularly important in tissue regeneration and mimicking vascular tissue in vitro. Numerous studies have described the effects of ECs on nanofibers made from a variety of synthetic polymer materials designed to mimic the extracellular matrix (ECM). However, little is known about maintaining the integrity of ECs in in vitro systems. Here we describe polycaprolactone nanofibrous membranes coated with collagen gel that overcome many limitations of conventional nanofibers used for engineering endothelia. We investigated cell-cell and cell-ECM junctional complexes using collagen-coated and conventional nanofibrous membranes. Conventional nanofibrous membranes alone did not form a monolayer with ECs, whereas collagen-coated nanofibrous membranes did. Several concentrations of collagen in the gel coating promoted the formation of cell-cell junctional complexes, facilitated the deposition of laminin, and increased the focal contact organization of ECs. These results suggest the possible use of collagen-coated nanofibrous membranes for vascular tissue engineering applications and a vascular platform for organ-on-a-chip systems.