Endothelial cell-cell contacts are essential for vascular integrity and physiology, protecting tissues and organs from edema and uncontrolled invasion of inflammatory cells. The vascular endothelial barrier is dynamic, but its integrity is preserved through a tight control at different levels. Inflammatory cytokines and G-protein-coupled receptor agonists, such as histamine, reduce endothelial integrity and increase vascular leakage. This is due to elevated myosin-based contractility, in conjunction with phosphorylation of proteins at cell-cell contacts. Conversely, reducing contractility stabilizes or even increases endothelial junctional integrity. Rho GTPases are key regulators of such cytoskeletal dynamics and endothelial cell-cell contacts. In addition to signaling-induced regulation, the expression of junctional proteins, such as occludin, claudins and vascular endothelial cadherin, also controls endothelial barrier function. There is increasing evidence that, in addition to protein phosphorylation, ubiquitylation (also known as ubiquitination) is an important and dynamic post-translational modification that regulates Rho GTPases, junctional proteins and, consequently, endothelial barrier function. In this Review, we discuss the emerging role of ubiquitylation and deubiquitylation events in endothelial integrity and inflammation. The picture that emerges is one of increasing complexity, which is both fascinating and promising given the clinical relevance of vascular integrity in the control of inflammation, and of tissue and organ damage.