Salivary secretory immunoglobulin A (S-IgA) antibodies act as the first line of defense against dental caries by blocking of adherence of Streptococcus mutans to tooth surfaces. This study focused on finding proper mucosal immunization route and delivery system to induce higher level of specific anti-S. mutans saliva S-IgA and inhibit dental caries in animal model. By immunizing rats with an anti-caries DNA vaccine, pCIA-P, via different mucosal routes, we found that intranasal (i.n.) immunization with pCIA-P/bupivacaine DNA complexes elicited the highest specific anti-S. mutans saliva S-IgA mucosal antibody responses compared with naked DNA and other routes. Correspondingly, rats immunized with pCIA-P/bupivacaine DNA complex via i.n. displayed the least carious lesions. Our findings suggested that DNA vaccination via intranasal immunization with bupivacaine delivery system be a promising approach against dental caries.