OBJECTIVE ECV304/C6 co-culture model is a widely used tool for BBB studies. However, cell source may influence the establishment of co-culture model and some C6 cells could damage the barrier integrity. Here, we established an ECV304 monoculture model and evaluated it in the respect of tightness, tight junction proteins and discriminative brain penetration. METHODS The tightness of ECV304 cell layers was evaluated by the measurement of permeability to hydrophilic marker Lucifer yellow. Immunofluorescence method was explored to detect the expression of tight junction proteins occludin, claudin-5 and ZO-1 in ECV304 cells. The discriminative brain penetration of the model was assessed by a permeability testing of compounds with different penetration rates, including digoxin, quinidine, and propranolol. RESULTS The ECV304 monolayers developed low permeability to Lucifer yellow (permeability coefficient: 0.31 ± 0.02 × 10-3 cm/min) and exhibited positive immunostaining of occludin, claudin-5 and ZO-1. The permeability coefficients of high permeable quinidine and propranolol across ECV304 cell layers were higher than that of low permeable digoxin by 3.6 and 2.8-fold, respectively. CONCLUSIONS The ECV304 monoculture model developed tight paracellular barrier and discriminated between compounds with different permeability, indicating it as a potential in vitro model for BBB permeability assessment.