Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition. Display omitted ► PV1 expression in vascular endothelium is required for survival ► PV1 is required for the formation of stomatal and fenestral diaphragms ► Lack of diaphragms in fenestrated endothelia causes vascular leak of plasma proteins ► Vascular leak results in severe hypoproteinemia and hypertriglyceridemia Plasmalema vesicle-associated protein (PV1) is critical for the formation of diaphragms in endothelial caveolae, fenestrae, and transendothelial channels. Using mice with loss and gain of PV1 function, Stan et al. show that the diaphragms of fenestrae are critical for the control of basal permeability, blood composition, and survival.