We previously reported that rabies virus strain CE(NiM), but not the parental Ni-CE strain, killed mice after intracerebral inoculation. CE(NiM) and Ni-CE are genetically identical except for two amino acids at positions 29 and 95 in the M protein. In this study, to identify which residue determines the pathogenicity, we examined pathogenicities of two Ni-CE mutants, CE(NiM29) and CE(NiM95), which were established by replacement of an amino acid residue at position 29 or 95 in the Ni-CE M protein with the corresponding residue of CE(NiM), respectively. We found that CE(NiM95), but not CE(NiM29), killed mice, indicating that the amino acid at position 95 in the M protein is the pathogenic determinant.