The endothelial layer of blood vessels controls the passage of cells and solutes from the blood into the surrounding tissue. Crucial for this regulation is the integrity of endothelial cell–cell junctions. Various molecular mechanisms control junctional integrity of the endothelial layer including GTPases, modulation of the actomyosin cytoskeleton and phosphorylation and dephosphorylation of junctional proteins. Several kinases and phosphatases have been identified that are good candidates for the regulation of the endothelial barrier function. For some of them, in vivo evidence has recently been presented that highlights their importance in either the regulation of vascular permeability or leukocyte extravasation. This review will summarize current knowledge about the regulation of endothelial junctions by kinases and phosphatases. In particular, the role of the endothelial specific phosphatase VE-PTP in the context of endothelial cell contact stability will be highlighted.