Endothelial cell behavior is regulated by subendothelial extracellular matrix (ECM). The ECM protein fibronectin (FN) is rare in healthy blood vessels but accumulates in disease accompanied by endothelial dysfunctions. Here, we report that excess assembly of FN disrupts key endothelial functions. We mimicked increased FN expression as in diseased stroma by providing exogenous FN basally in a Transwell insert and found dose-dependent upregulation of subendothelial FN matrix assembly. Taking advantage of discontinuous matrix assembly by endothelial cells, we show correlations between regional increases in FN matrix and disruptions in endothelial cell morphology, VE-cadherin junctions, and the cell cycle, all of which were not changed in FN-deficient regions of the monolayer. These changes affected endothelial barrier function with increased monolayer permeability exposing basal regions of high FN matrix and permitting FN-dependent adhesion of MDA-MB-231 tumor cells from the apical side of the monolayer. FN matrix accumulation was quick and increases in FN matrix preceded all other changes in the endothelium. Therefore, subendothelial accumulation of FN matrix is a cause, not an effect, of endothelial monolayer disorganization and leakiness. Regulating FN accumulation in the subendothelial space could be an important target for controlling progression of fibrosis and related diseases.