Administration of pigment epithelium-derived factor (PEDF) inhibits advanced glycation end products-elicited retinal vascular hyperpermeability, as well as cold injury-induced brain oedema in rats. However, the underlying molecular mechanism by which PEDF blocks the hyperpermeability in vivo is not fully understood. This study investigated whether PEDF could inhibit vascular endothelial growth factor (VEGF)-induced vascular hyperpermeability both in vitro and in vivo. The Miles assay revealed that, after intradermal injection of VEGF in nude mice, simultaneous administration of PEDF inhibited vascular hyperpermeability in a dose-dependent manner. The in vitro permeability assay also showed that PEDF blocked the VEGF-induced barrier dysfunction in endothelial cells. These results demonstrated that PEDF could inhibit the VEGF-induced vascular hyperpermeability both in vitro and in vivo, and suggest that PEGF may be suitable to be considered as a novel therapeutic agent for various vasopermeable disorders in which VEGF is involved.