Live attenuated strains of the Japanese encephalitis (JE) virus are known to form in various cultured cells. In this study, we selected attenuated JE virus variants by passing the parent virus strains once through Neuro-2a cells, showing that the selection intensity ranged 2-1000-fold after infection for 3d. The selection of attenuated variants appeared in association with mutations on the envelope (E) glycoprotein. This is likely determined by the differential binding abilities of specific E proteins with highly sulfated glycosaminoglycans on the cell surface. A plaque-purification method showed that Neuro-2a-selected variants were usually less neurovirulent, leading to a longer survival time of intracerebrally inoculated mice. Specific selected variants (mostly with the small-plaque phenotype) were shown to be more efficient at replication in Vero cells and less virulent, particularly those with substitution of the E-138 (Glu-->Lys) residue. As a result, most, if not all, low-virulent variants were generated in a relatively short time, all which have the potential to be live-attenuated vaccine candidates of the JE virus.