In humans, heparin‐binding protein (HBP) and the potent chemotactic lipid leukotriene B₄ (LTB₄) are important mediators of innate immune reponses. Here we show that human neutrophils (PMNs) challenged with LTB₄ (30 s to 5 min) release HBP as determined by Western blot analysis. This response peaks at 100 nM of agonist and is mediated by the BLT1 receptor. Proteinphosphatase‐1 (30 μM) and wortmannin (0.5 μM) block the LTB₄‐mediated HBP release from PMNs, which suggests involvement of the 1‐phosphatidylinositol 3‐kinase intracellular pathway during degranulation. Furthermore, postsecretory supernatants from LTB₄‐stimulated PMNs induce intracellular calcium mobilization in endothelial cells in vitro and increase in vascular permeability in vivo, as assessed in a mouse model of pleurisy. Selective removal of HBP from the supernatant significantly reduces these activities attributing a key role to HBP in the LTB₄‐induced change in vascular permeability. This lipid‐protein axis could offer novel opportunities for pharmacological intervention in key steps of the vascular response to inflammation.—Di Gennaro, A., Kenne, E., Wan, M., Soehnlein, O., Lindbom, L., Haeggstrom, J.Z. Leukotriene B4‐induced changes in vascular permeability are mediated by neutrophil release of heparinbinding protein (HBP/CAP37/azurocidin). FASEB J. 23, 1750–1757 (2009)