Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference-based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.