We have successfully fabricated a human microvascular endothelial model with high endothelial barrier function by coculturing foreskin derived HMVECs and HDFs via a collagen vitrigel membrane (CVM) (Uzu & Takezawa, 2020). To develop a more versatile model, this study aimed to fabricate a culture model of hCMEC/D3 cells (a human cortex microvascular endothelial cell line) in a CVM chamber. The endothelial barrier function was assessed by measurement of transendothelial electrical resistance (TEER) and permeability of 4 model compounds (Mw: 376, 40,000, 10,000, and 40,000) with or without 100 µM histamine treatment. The phase-contrast microscopic observation of the original hCMEC/D3 cells revealed a heterogeneous population of cobblestone- and spindle-shaped cells. Then, we isolated uniform cobblestone-shaped clone A from the original cells by limiting dilution. TEER value of a monolayer of clone A cultured in a CVM chamber reached 30 Ω･cm². Clone A retained its endothelial-like morphology and endothelial barrier function even after 30 times cell division. Histamine treatment caused 70% decrease of TEER value from the initial value, which was prevented by pretreatment with Y-27632. Interestingly, the permeability of the compounds with a molecular weight of not 376 and 40,000 but 4,000 and 10,000 was selectively increased by histamine. In conclusion, a microvascular endothelial model of clone A isolated from hCMEC/D3 cells cultured in a CVM chamber is useful for predicting histamine-induced microvascular hyperpermeability with high reproducibility.