Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin‐α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.—Wei, H., Sundararaman, A., Dickson, E., Rennie‐Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome. FASEB J. 33, 12277‐12287 (2019). http://www.fasebj.org