Japanese encephalitis is the major cause of viral encephalitis in Southeast Asia, which is caused by Japanese encephalitis virus (JEV), a Flavivirus belonging to the family Flaviviridae. JEV is transmitted through an infected mosquito bite during ingestion of blood meal. Most of the infected individuals are asymptomatic; however, some of the individuals may present symptoms of acute encephalitis syndrome (AES). It has been shown that the BBB present in the cerebrum is the initial site of JEV entry into the CNS. JEV primarily affects hippocampus, thalamus, medulla oblongata, and substantia nigra upon neuroinvasion. However, brain stem, cerebellum, cerebral white matter, and cerebral cortices are less likely to be affected. As JEV enters the brain via crossing the blood-brain barrier, it initiates the apoptotic proteins in brain microvascular endothelial cells. Following entry, JEV induces apoptosis in neurons. Remarkably, dopaminergic neuron-rich brain regions along with the midbrain and thalamus are targeted mostly during JEV infection. JEV induces microglia cell activation that results in the release of proinflammatory mediators that leads to neuronal cell death and inflammation of the brain. Astrocytes, on the other hand, are also targeted by JEV, which results in the induction of proinflammatory cytokines. Comparatively, oligodendrocyte cells are less affected by JEV. However, there is little understanding of the mechanism of blood-brain barrier breaching, major CNS target cells, and molecular mechanisms of JEV neuropathogenesis. Therefore, this chapter focuses on the mechanism of crossing the blood-brain barrier, major CNS target cells, and impact of JEV on these cell types.