Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date， Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide， which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report， the generation of Ad5-CEAB， a recombinant adenovirus expressing Mtb antigens of CFP10， ESAT6， Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB， mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response， particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines， IFN-γ， TNF-α， IL-2 and IL-12. In addition， the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore， an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice， suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo， which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.