Life-threatening inflammatory conditions such as acute respiratory distress syndrome or sepsis often go hand in hand with severe vascular leakage. During inflammation, endothelial cell integrity and intact barrier function are crucial to limit leukocyte and plasma extravasation. Prostaglandin D (PGD) is a potent inflammatory lipid mediator with vasoactive properties. Previous studies suggest that PGD is involved in the regulation of endothelial barrier function; however, it is unclear whether this is also true for primary human pulmonary microvascular endothelial cells. Furthermore, as PGD is a highly promiscuous ligand, we set out to determine which receptors are important in human pulmonary endothelial cells. In the current study, we found that PGD and the DP1 agonist BW245c potently strengthened pulmonary and dermal microvascular endothelial cell barrier function and protected against thrombin-induced barrier disruption. Yet surprisingly, these effects were mediated only to a negligible extent via DP1 receptor activation. In contrast, we observed that the EP4 receptor was most important and mediated the barrier enhancement by PGD and BW245c. Stimulation with PGE or PGD reduced AKT phosphorylation which could be reversed by prior blockade of EP4 receptors. These data demonstrate a novel mechanism by which PGD may modulate inflammation and emphasizes the role of EP4 receptors in human endothelial cell function.Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.