Multicellular spheroidal (MCS) culture system is a promising culture method for development of a highly functional in vitro BBB model, which is expected to be a useful tool for central nervous system (CNS) drug development studies. By taking advantage of their scalability and functionality, immortalized cells are useful for development of such MCS-BBB models, and we have recently established human conditionally immortalized brain microvascular endothelial cells (HBMEC/ci18), astrocytes (HASTR/ci35), and pericytes (HBPC/ci37). Therefore, utilizing these cells, we aimed to develop human immortalized cell-based MCS-BBB models (hiMCS-BBB) and characterize their BBB properties. The cells in a V-bottom well were self-assembled into a spheroid, and HBMEC/ci18 formed the outer monolayer of the spheroids. Furthermore, while most HASTR/ci35 accumulated in the core, the majority of HBPC/ci37 aligned along the inner side of HBMEC/ci18. Next, we examined the BBB properties of hiMCS-BBB. We found that hiMCS-BBB showed 0.28-fold lower FITC-dextran (5 kDa) permeability compared with hiMCS-BBB without HBMEC/ci18. These results suggest that HBMEC/ci18 forms a functional barrier in hiMCS-BBB. Collectively, we have developed the hiMCS-BBB that exhibits BBB properties. The hiMCS-BBB holds the potential as a new in vitro experimental tool that will contribute to CNS drug development.