Genotype V (GV) Japanese encephalitis virus (JEV) has emerged in Korea and China since 2009. Recent findings suggest that current Japanese encephalitis (JE) vaccines may reduce the ability to induce neutralizing antibodies against GV JEV compared to other genotypes. This study sought to produce a novel live attenuated JE vaccine with a high efficacy against GV JEV. Genotype I (GI)-GV intertypic recombinant strain rJEV-EXZ0934-M41 (EXZ0934), in which the E region of the GI Mie/41/2002 strain was replaced with that of GV strain XZ0934, was introduced with the same 10 attenuation substitutions in the E region found in the live attenuated JE vaccine strain SA 14-14-2 to produce a novel mutant virus rJEV-EXZ/SA14142m-M41 (EXZ/SA14142m). In addition, another mutant rJEV-EM41/SA14142m-M41 (EM41/SA14142m), which has the same substitutions in the Mie/41/2002, was also produced. The neuroinvasiveness and neurovirulence of the two mutant viruses were significantly reduced in mice. The mutant viruses induced neutralizing antibodies against GV JEV in mice. The growth of EXZ/SA14142m was lower than that of EM41/SA14142m. In mouse challenge tests, a single inoculation with a high dose of the mutants blocked lethal GV JEV infections; however, the protective efficacy of EXZ/SA14142m was weaker than that of EM41/SA14142m in low-dose inoculations. The lower protection potency of EXZ/SA14142m may be ascribed to the reduced growth ability caused by the attenuation mutations.