Human adenovirus (HAdV) type 40 in species F (HAdV-F40) and HAdV-F41 represent the third most prevalent causative agents of non-bacterial acute gastroenteritis in infants and young children, following norovirus and rotavirus. Despite their significant contribution to global child morbidity, vaccines to preemptively combat these viruses remain elusive. In this study, we scrutinize the potential for cross-neutralization between HAdV-F40 and HAdV-F41 using the knob protein of the fiber-2 protein immunized sera. To this end, we immunized female BALB/c mice with synthetically produced knob proteins of the fiber-2 protein from HAdV-F40 and HAdV-F41. Subsequently, we implemented a series of assays to evaluate the results, including enzyme-linked immunosorbent, micro-neutralization, immunofluorescence, and quantitative polymerase chain reaction. We found that HAdV-F40-knob and HAdV-F41-knob immunized sera could effectively neutralize HAdV-F40 and HAdV-F41, indicating a mutual cross-neutralizing effect. Notably, the serum immunized with HAdV-F40-knob demonstrated a stronger neutralization effect, suggesting the potential to develop a subunit vaccine that can simultaneously counteract both viruses. Our findings underscore the potential of knob protein immunization in evoking a cross-neutralizing antibody response between HAdV-F40 and HAdV-F41. This suggests a promising avenue for developing subunit vaccines against HAdV-F40 and HAdV-F41 and provides a novel perspective on the potential of neutralizing antibodies to protect against these two types of HAdV.