The passage of leukocytes out of the blood circulation and into tissues is necessary for the normal inflammatory response, but also occurs inappropriately in many pathological situations. This process is limited by the barrier presented by the junctions between adjacent endothelial cells which line blood vessels. Here we show that activation of the Rap1 GTPase in endothelial cells accelerates de novo assembly of endothelial cell-cell junctions and increases the barrier function of endothelial monolayers. In contrast, depressing Rap1 activity by expressing Rap1GAP leads to disassembly of these junctions and increases their permeability. We also demonstrate that endogenous Rap1 is rapidly activated at early stages of junctional assembly, confirming the involvement of Rap1 during junctional assembly. Intriguingly, elevating Rap1 activity selectively within endothelial cells decreases leukocyte transendothelial migration, while inhibiting Rap1 activity by expression of Rap1GAP increases leukocyte transendothelial migration, providing physiological relevance to our hypothesis that Rap1 augments barrier function of interendothelial cell junctions. Furthermore, these results suggest that Rap1 may be a novel therapeutic target for clinical conditions where an inappropriate inflammatory response leads to disease.