Vascular endothelial cells contain unique rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs), which contain the hemostatic protein Von Willebrand factor (VWF) and a cocktail of angiogenic and inflammatory mediators. We have shown that the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormone-evoked WPB exocytosis. Using a non-biased proteomic screen for targets for Slp4-a we now identify syntaxin binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells. Co-immunoprecipations showed that STXBP1 interacts with syntaxin-2 and -3, but not syntaxin-4. siRNA-mediated silencing of STXBP1 expression impaired histamine- and forskolin-induced VWF secretion. To further substantiate the role of STXBP1 we isolated blood outgrowth endothelial cells (BOECs) from an early infantile epileptic encephalopathy type 4 (EIEE4) patient carrying a de novo mutation in STXBP1. STXBP1 haploinsufficient EIEE4 BOECs contained similar numbers of morphologically normal WPBs compared to control BOECs of healthy donors, however, EIEE4 BOECs displayed significantly impaired histamine- and forskolin-stimulated VWF secretion. Based on these findings, we propose that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1, thereby controlling the release of vaso-active substances in the vasculature.