In our previous study, we have demonstrated in the context of WNV-ΔNS1 vaccine (a replication-defective West Nile virus (WNV) lacking NS1) that the NS1 trans-complementation system may offer a promising platform for the development of safe and efficient flavivirus vaccines only requiring one dose. Here, we produced high titer (107 IU/ml) replication-defective Japanese encephalitis virus (JEV) with NS1 deletion (JEV-ΔNS1) in the BHK-21 cell line stably expressing NS1 (BHKNS1) using the same strategy. JEV-ΔNS1 appeared safe with a remarkable genetic stability and high degrees of attenuation of in vivo neuroinvasiveness and neurovirulence. Meanwhile, it was demonstrated to be highly immunogenic in mice after a single dose, providing similar degrees of protection to SA14-14-2 vaccine (a most widely used live attenuated JEV vaccine), with healthy condition, undetectable viremia and gradually rising body weight. Importantly, we also found JEV-ΔNS1 induced robust cross-protective immune responses against the challenge of heterologous West Nile virus (WNV), another important member in the same JEV serocomplex, accounting for up to 80% survival rate following a single dose of immunization relative to mock-vaccinated mice. These results not only support the identification of the NS1-deleted flavivirus vaccines with a satisfied balance between safety and efficacy, but also demonstrate the potential of the JEV-ΔNS1 as an alternative vaccine candidate against both JEV and WNV challenge.