The blood-brain barrier (BBB), connected by interendothelial tight junctions, hinders the passage of substances into central nerve system. To mimic in vivo BBB conditions for pathological and pharmacological studies, the immortalized cell lines were developed. However, the brain endothelial cell lines often demonstrate insufficient tightness and thus the non-cerebral cell lines such as ECV304 cells are utilized as a BBB-mimicking tool. In this study, the expression of tight junction proteins occludin, claudin-5 and ZO-1 were confirmed in ECV304 cells by RT-PCR and Western-blot methods, and the immunofluorescence revealed the localization of three tight junction proteins along cell border, indicating the formation of tight junctions among ECV304 cells. The ECV304 monoculture model on polyethylene terephthalate (PET) membrane transwell inserts demonstrated higher TEER and lower permeability to hydrophilic marker lucifer yellow than that on polyester inserts. In addition, the permeability ratio of lipophilic marker propranolol to lucifer yellow was higher on PET model than that on polyester model. These results showed that the PET model of ECV304 cells developed tighter barrier and better discriminated between paracellular and transcellular flux than polyester model, suggesting its potential for passive transport studies.