A central function of the vascular endothelium is to serve as a barrier between the blood and the surrounding tissue of the body. At the same time, solutes and cells have to pass the endothelium to leave or to enter the bloodstream to maintain homeostasis. Under pathological conditions, for example, inflammation, permeability for fluid and cells is largely increased in the affected area, thereby facilitating host defense. To appropriately function as a regulated permeability filter, the endothelium uses various mechanisms to allow solutes and cells to pass the endothelial layer. These include transcellular and paracellular pathways of which the latter requires remodeling of intercellular junctions for its regulation. This review provides an overview on endothelial barrier regulation and focuses on the endothelial signaling mechanisms controlling the opening and closing of paracellular pathways for solutes and cells such as leukocytes and metastasizing tumor cells.