PGE2 is a major prostanoid that regulates inflammation by stimulating EP1-4 receptors. However, how PGE2 induces an initial inflammatory response to vascular hyper-permeability remains unknown. Here we investigated the role of the PGE2 -EP receptor signal in modulating vascular permeability both in vivo and in vitro.We used a modified Miles assay and intravital microscopy to examine vascular permeability in vivo. Endothelial barrier property was assessed by measuring transendothelial electrical resistance (TER) in vitro.Local administration of PGE2 , an EP2 or EP4 receptor agonist into FVB/NJcl mouse ear skin caused vascular leakage, indicated by dye extravasation. Intravital microscopy and laser Doppler blood-flow imaging revealed that these treatments dilated peripheral vessels and increased local blood flow. Pretreatment with the vasoconstrictor phenylephrine inhibited the PGE2 -induced blood flow increase and vascular leakage. In contrast to the EP2 and EP4 receptor agonists, administration of an EP3 receptor agonist suppressed vascular leakage without altering vascular diameter or blood flow. In isolated HUVECs, the EP3 receptor agonist elevated TER and blocked thrombin-induced dextran passage. Inhibiting PKA restored the hypo-permeability induced by the EP3 receptor agonist.Activation of the PGE2 -EP2 or -EP4 receptor signal induces vasodilatation in mural cells, resulting in increased local blood flow and hyper-permeability. In contrast, activation of the PGE2 -EP3 receptor signal induces a cAMP-dependent enhancement of the endothelial barrier, leading to hypo-permeability. We provide the first evidence that endothelial cells and mural cells cooperate to modulate vascular permeability.