Background: Human adenoviruses (HAdVs) frequently cause common respiratory or gastrointestinal infections among children, adults, individuals with immune deficiencies and other vulnerable populations with varying degree of symptoms, ranging from mild to server, and in some cases, even fatalities. Despite the significant clinical impact of HAdVs, there is currently no approved vaccine available. Methods: This study explores the potential of the adenovirus type 5 fiber knob (Ad5-FK) to stimulate the production of Ad-specific neutralizing antibodies and T-cell responses in mice. Based in structure predictions, we firstly expressed Ad5-FK in E. coli and confirmed the assembly of FK into trimerc form. After testing the binding capability of the trimeric FK to susceptible cells, the immunogenicity of the protein, in combination with the c-di-AMP adjuvant was assessed in BALB/c mice. Results: The purified Ad5-FK exhibited self-trimerization and maintained correct conformation akin to the authentic FK structure. This facilitated effective binding to susceptible HEK293 cells. Notably, the protein demonstrated significant inhibition of HEK293 cells infection by rAd5-GFP. Immunization of BALB/c mice with Ad5-FK or Ad5-FK mixed with c-di-AMP yielded FK-specific antibodies with potent neutralization capacity. Significantly, Ad5-FK was found to elicit a vigorous CD4+ T-cell response in the immunized mice. Conclusion: Our findings underscore the efficacy of FK-based vaccine in eliciting anti-Ad humoral immune response and CD4 T-cell immune reactions essential for protection against viral infections.