The vascular endothelium forms a barrier between the circulation and the interstitium, and changes in vascular permeability are very important during vascular repair and inflammatory conditions. The regulation of the endothelial barrier depends on actomyosin-related cell contractility as well as the integrity of cell-cell junctions, such as tight and adherens junctions. The present review will introduce the basic pathways involved in the regulation of the endothelial barrier as well as important participating signaling players, such as the small GTPases RhoA, Rac and Rap1. In this context, we will attempt to analyze and interpret the recent evidence pointing to the role of the family of junctional adhesion molecules (JAMs) in modulating the pathways involved in the regulation of the endothelial barrier.