Endothelial cells (ECs) form a monolayer that serves as a barrier between the blood and the underlying tissue. ECs tightly regulate their cell-cell junctions, controlling the passage of soluble materials and immune cells across the monolayer barrier. We studied the role of N-WASP, a key regulator of Arp2/3 complex and actin assembly, in EC monolayers. We report that N-WASP regulates endothelial monolayer integrity by affecting the organization of cell junctions. Depletion of N-WASP resulted in an increase in transendothelial electrical resistance, a measure of monolayer integrity. N-WASP depletion increased the width of cell-cell junctions and altered the organization of F-actin and VE-cadherin at junctions. N-WASP was not present at cell-cell junctions in monolayers under resting conditions, but it was recruited following treatment with sphingosine-1-phosphate. Taken together, our results reveal a novel role for N-WASP in remodeling EC junctions, which is critical for monolayer integrity and function.