Human adenovirus type 4 (HAdV-4) is an epidemic virus that contributes to serious acute respiratory disease (ARD) in both pediatric and adult patients. However, no licensed drug or vaccine is currently available to the civilian population. The identification of neutralizing epitopes of HAdV-4 should allow the development of a novel antiviral vaccine and a novel gene transfer vector, and an effective neutralizing monoclonal antibody (MAb) will be useful in developing appropriate antiviral drugs. In this study, we report that MAb MN4b shows strong neutralizing activity against HAdV-4. MN4b recognizes a conformational epitope (418AGSEK422) within hypervariable region 7 (HVR7). Mutations within this site permitted HAdV-4 mutants to escape neutralization by MN4b and to resist neutralization by animal and human anti-HAdV-4 sera. A recombinant virus, rAd3-A4R7-1, containing the identified neutralizing epitope in the HVR7 region of HAdV-3 hexon, successfully induced antiserum that inhibited HAdV-4 infection. These results indicate that a small surface loop of HAdV-4 hexon is a critical neutralization epitope for this virus. The generation of MN4b and the identification of this neutralizing epitope may be useful in developing therapeutic treatment, a subunit vaccine, and a novel vector that can escape preexisting neutralization for HAdV-4.