Endothelial monolayer permeability is regulated by actin dynamics and vesicular traffic. Recently, ubiquitination was also implicated in the integrity of quiescent endothelium, as it differentially controls the localization and stability of adhesion- and signaling proteins. We found that inhibition of E1 ubiquitin ligases induces a rapid, reversible loss of integrity in quiescent, primary human endothelial monolayers, accompanied by increased F-actin stress fibers and the formation of intercellular gaps. Concomitantly, total protein and activity of the actin-regulating GTPase RhoB, but not its close homologue RhoA, increase ~10-fold in 5-8 h. The depletion of RhoB, but not of RhoA, the inhibition of actin contractility and the inhibition of protein synthesis all significantly rescue the loss of cell-cell contact induced by E1 ligase inhibition. Our data suggest that in quiescent human endothelial cells, the continuous and fast turnover of short-lived proteins that negatively regulate cell-cell contact, is essential to preserve monolayer integrity.