Endothelial barrier dysfunction, which is a serious problem that occurs in various inflammatory conditions, permits extravasation of serum components into the surrounding tissues, leading to edema formation and organ failure. Pigment epithelium-derived factor (PEDF), which is a major endogenous antagonist, has been implicated in diverse biological process, but its role in endothelial barrier dysfunction has not been defined. To assess the role of PEDF in the vasculature, we evaluated the effects of exogenous PEDF using human umbilical vein endothelial cells (HUVECs) in vitro. Our results demonstrated that exogenous PEDF activated p38/MAPK signalling pathway in a dose- and time-dependent manner and induced vascular hyperpermeability as measured by the markedly increased FITC-dextran leakage and the decreased transendothelial electrical resistance (TER) across the monolayer cells, which was accompanied by microtubules (MTs) disassembly and F-actin rearrangement. However, the aforementioned alterations can be arrested by the application of low concentration of p38/MAPK inhibitor SB203580. These results reveal a novel role for PEDF as a potential vasoactive substance in inducing hyperpermeability. Furthermore, our results suggest that PEDF and p38/MAPK may serve as therapeutic targets for maintaining vascular integrity.