Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that JEV getsaccess to the central nervous system (CNS) as a consequence of a precedinginflammatory process which leads to the blood-brain barrier (BBB) disruption and viral neuroinvasion. However, what happens at early times ofJEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in cellulo humanBBB model consisting of hCMEC/D3 human endothelial cells cultivated on permeable insertsabove SK-N-SH human neuroblastoma cells. Usingthis system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disruptingit at early times post-addition. Furthermore, this BBB model was able to discriminate between the virulent RP9 and the vaccine SA14-14-2 strains, as demonstrated by the presence of almost 10 times more RP9 infectious particles that crossed the BBB than SA14-14 particlesat a high MOI. Besidescontributingtothe understanding of early events in JEV neuroinvasion,thisin celluloBBB model represents asuitable anduseful systemto studythe viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crossesthe BBBat early times of neuroinvasion