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 investigate the potential for cross-neutralization between HAdV-F40 and HAdV-F41 using immune sera with the short fiber knob (SFK). We implemented a series of assays to evaluate the responses, including enzyme-linked immunosorbent, micro-neutralization, immunofluorescence, and quantitative polymerase chain reaction. Our results demonstrate that immune sera with HAdV-F40 SFK or HAdV-F41 SFK could effectively neutralize both HAdV-F40 and HAdV-F41, indicating a mutual cross-neutralizing effect. Notably, the immune sera with HAdV-F40 SFK demonstrated a stronger neutralization effect, suggesting the potential to develop a subunit vaccine that can simultaneously counteract both viruses. These findings underscore the potential of SFK 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.