Although the endothelium is an extremely thin single-cell layer, it performs exceedingly well in preventing blood fluids from leaking into the surrounding tissues. However, specific pathological conditions can affect this cell layer, compromising the integrity of the barrier. Vascular leakage is a hallmark of many cardiovascular diseases and despite its medical importance, no specialized therapies are available to prevent it or reduce it. Small guanosine triphosphatases (GTPases) of the Rho family are known to be key regulators of various aspects of cell behavior and studies have shown that they can exert both positive and negative effects on endothelial barrier integrity. Moreover, extracellular matrix stiffness has now been implicated in the regulation of Rho-GTPase signaling, which has a direct impact on the integrity of endothelial junctions. However, knowledge about both the precise mechanism of this regulation and the individual contribution of the specific regulatory proteins remains fragmentary. In this review, we discuss recent findings concerning the balanced activities of Rho-GTPases and, in particular, aspects of the regulation of the endothelial barrier. We highlight the role of Rho-GTPases in the intimate relationships between biomechanical forces, microenvironmental influences and endothelial intercellular junctions, which are all interwoven in a beautiful filigree-like fashion.