Injury of vascular endothelial barrier function is implicated in several pathophysiological processes. The integrity of vascular endothelium is regulated by cytoskeleton and cell-cell junctions. Small guanosine triphosphatases of the Rho family (Rho GTPases) are known to play a central role in vascular endothelial barrier function. It has been reported that RhoA, Rac1, Cdc42 and RhoB are involved and they exert both positive and negative effect on endothelial barrier integrity, depending on their subcellular location. When inflammatory factors such as thrombin attack the vascular endothelial cells, GEF of RhoA will be widely distributed throughout the cells. Thus, activated RhoA causes aggregation of F-actin fibers in a short time and disrupts the vascular endothelial barrier, a process named acute cell contraction. However, RhoA may also induce the production and maturation of intercellular junctions in new cells. Rac1 and Cdc42 help to maintain the integrity of vascular endothelial barrier at the resting state. They cause the phosphorylation of LIM kinase and inhabitation of cofilin, resulting in less remodeling of cytoskeletal in the vascular endothelial cells. On the other hand, Cdc42 can translocate to the cortex rapidly after a stimulation, where Cdc42 will activate the myosin Ⅱ and promote the reorganization of adjective junction to facilitate the recovery of vascular endothelial barrier. In this review, we overviewed how Rho GTPases regulate the vascular endothelial barrier integrity.