The vascular endothelium operates in a highly polarized environment, but to date there has been little exploration of apicobasal polarization of its signaling. We show that VEGF-A, histamine, IGFBP3, and LPA trigger unequal endothelial responses when acting from the circulation or the parenchymal side at blood-neural barriers. For VEGF-A, highly polarized receptor distribution contributed to distinct signaling patterns: VEGFR2, which was found to be predominantly abluminal, mediated increased permeability via p38; in contrast, luminal VEGFR1 led to Akt activation and facilitated cytoprotection. Importantly, such differential apicobasal signaling and VEGFR distribution were found in the microvasculature of brain and retina but not lung, indicating that endothelial cells at blood-neural barriers possess specialized signaling compartments that assign different functions depending on whether an agonist is tissue or blood borne.