The current live rabies vaccine strain SAG2 is attenuated by only one mutation (Arg-to-Glu) at position 333 in the glycoprotein (G333), generating a potential risk of the emergence of a pathogenic revertant by a back mutation at this position during viral propagation in the body. To circumvent this risk, it is desirable to generate a live vaccine strain stably attenuated by multiple mutations. However, little is known about attenuating mutations other than that at G333. In this study, we found that amino acid substitutions at positions 273 and 394 in the nucleoprotein (N273/394) (Phe-to-Leu and Tyr-to-His, respectively) attenuated the pathogenicity of the oral live vaccine strain ERA, which has a virulent-type Arg at G333. Then we generated ERA-N273/394-G333 strain attenuated by the combination of the above attenuating mutations at G333 and N273/394, and we checked its safety and immunogenicity. ERA-N273/394-G333 strain did not cause any symptoms in adult mice after intracerebral inoculation, indicating a low level of residual pathogenicity of this strain. Also, we found that the risk of the emergence of a pathogenic revertant of ERA-N273/394-G333 strain was lower than that of the ERA-G333 strain, which has a single attenuating mutation at G333. Furthermore, intramuscular inoculation of this strain induced protective immunity in adult mice against lethal rabies infection. These results indicate that ERA-N273/394-G333 strain is a promising candidate for a live rabies vaccine strain with a high level of safety.