Hepatitis E virus (HEV) genotype 4 was originally identified in China. Its neutralization antigenic epitopes have not been characterized. Recently, we identified a neutralizing monoclonal antibody (mAb) 1G10, which was generated following immunization of mice with p166Chn, a recombinant protein comprising 464-629 amino acids (aa) of the HEV genotype 4 capsid protein. In this study, a panel of 22 N- and/or C-terminal truncated and 6 site-directed mutated p166Chn proteins were prepared. Only those N- or C-terminal truncated proteins containing the region 477-613 aa could react with the mAb 1G10, suggesting the neutralization epitope of HEV genotype 4 is located between aa477 and aa613. However, a both N- and C-terminal truncated protein, pN477-C613, neither reacted to 1G10 nor elicited neutralizing antibodies in mice, while another both terminal truncated protein, pN472-C617, did, suggesting the flanking regions of the pN477-C613 could help to stabilize and allow presentation of the neutralization epitope to the immune system. Substituting Leu477 and/or Leu613 with the polar, uncharged threonine (Thr) caused ≥ 50% reduction of the mutants' immunoreactivity to 1G10, whereas replacement by hydrophobic phenylalanine (Phe) made little impact on the immunoreactivity, revealing functional associations between hydrophobicity of aa at positions 477 and 613 and the antigenicity of p166Chn. These data suggested Leu477 and Leu613 are critical in forming the neutralization epitope of HEV genotype 4. Its neutralization antigenic epitopes have not been characterized. Recently, we identified a neutralizing monoclonal antibody (mAb) 1G10, which was generated following immunization of mice with p166Chn, a recombinant protein comprising 464-629 amino acids (aa) of the HEV genotype 4 capsid protein. In this study, a panel of 22 N- and / or C-terminal truncated and 6 site-directed mutated p166 John proteins were prepared. Only those N- or C suggesting that the neutralization epitope of HEV genotype 4 is located between aa477 and aa 613. However, a both N- and C-terminal truncated protein, pN477-C613, neither reacted to 1G10 nor elicited neutralizing antibodies in mice, while another both truncated protein, pN472-C617, did, suggesting the flanking regions of the pN477-C613 could help to stabilize and allow presentation of the neut ralization epitope to the immune system. Substudy Leu477 and / or Leu613 with the polar, uncharged threonine (Thr) caused ≥50% reduction of the mutants' immunoreactivity to 1G10, revealing functional associations between hydrophobicity of aa at positions 477 and 613 and the antigenicity of p166Chn. These data suggest that Leu477 and Leu613 are critical in forming the neutralization epitope of HEV genotype 4.