The re-emerging human adenovirus (HAdV) types 3, 7, 14, and 55 of species B have caused severe or even fatal acute respiratory disease. Therefore, the development of multivalent vaccines against HAdV types 3, 7, 14, and 55 remains an important goal. In our previous study, we identified a cross-neutralizing epitope that induced broadly reactive monoclonal neutralizing antibodies against the knob proteins of HAdV types 7, 11, 14, and 55. To study the immunogenicity of HAdV species B knob proteins, we evaluated humoral immune responses to the knob proteins in vivo and in vitro. We found that the knob proteins elicited robust binding and neutralizing antibody responses after three immunizations of mice. In addition, mouse antisera raised against the knob proteins exhibited cross-neutralizing activity against original species B members. Furthermore, immunization with a mix of HAdV-3, 7, and 55 knob proteins protected Chinese tree shrews against an experimental HAdV challenge. Our results provide insight into the immunogenicity of HAdV species B knob proteins.IMPORTANCEHuman adenovirus (HAdV) species B are common pathogens causing severe pneumonia in children, and there is currently no vaccine available. Because there are many HAdV species B types, developing broad-spectrum vaccines against HAdV species B is an important research goal. Our study revealed that immunization with recombinant HAdV species B knob proteins effectively elicited cross-neutralizing antibody responses against original species B members with protective immunity. This study provides a novel insight into the immunogenicity of HAdV species B knob proteins.