Japanese encephalitis (JE) is a very severe disease characterized by high fatality rates and the development of permanent behavioral, psychiatric, and neurological sequelae among survivors. Japanese encephalitis virus (JEV), a flavivirus, is responsible for JE. In Asia, Genotype I (GI) has emerged as the dominant strain, replacing Genotype III (GIII). However, no clinically approved drug is available to treat JEV infection, and currently available commercial vaccines derived from JEV GIII strains provide only partial protection against GI. Utilizing a reverse genetics system, this study attempted to produce a novel chimeric JEV strain with high efficacy against JEV GI. Accordingly, a GI/GIII intertypic recombinant strain, namely SA14-GI env, was generated by substituting the E region of the GIII SA14-14-2 strain with that of the GI strain, K05GS. The neurovirulence of the mutant virus was significantly reduced in mice. Analysis of the immunogenicity of the chimeric virus revealed that it induced neutralizing antibodies against JEV GI in mice, and the protective efficacy of SA14-GI env was higher than that of SA14-14-2. These findings suggest that SA14-GI env may be a safe and effective live-attenuated vaccine candidate against JEV GI.