Respiratory mucosal immunization is an effective immunization strategy against tuberculosis (TB), and effective mucosal vaccines require adjuvants that can promote protective immunity without deleterious inflammation. Mucosal BCG (Bacille Calmette-Guerin) is effective, but it causes a severe inflammatory response in the lung. A novel less cytotoxic mucosal vaccine AH-PB containing (Mtb) cell surface antigens Ag85A and HspX (AH), as well as polyinosinic-polycytidylic acid (Poly IC) and bovine neutrophil β-defensin-5 (B5) adjuvants were prepared, with the overarching goal of protecting against TB. Then, the immunogenicity and protective efficacy of these vaccines via the intranasal route were evaluated in a mouse model. Results showed that intranasal AH-PB promoted tissue-resident memory T cells (T) development in the lung, induced antigen-specific antibody response in airway, provided protection against (), conferred better protection than parenteral BCG in the later stage of infection, and boosted the protective immunity generated by BCG in mice. Moreover, both B5 and Poly IC were indispensable for the protection generated by AH-PB. Furthermore, intranasal immunization with AH-B5 fusion vaccines also provided similar protection against compared to AH-PB. Collectively, B5-based TB vaccine via the intranasal route is a promising immunization strategy against bovine TB, and this kind of immunization strategy may be applied to human TB vaccine development. These findings highlight the potential importance of B5 as a mucosal adjuvant used in TB vaccines or other respiratory disease vaccines.