We have previously reported that a targeted anti-caries DNA plasmid pGJA-P/VAX which was constructed against the antigenic determinants of Streptococcus mutans (S. mutans) successfully induced antibody responses in mice and monkeys. The present study explored the protective efficacy of pGJA-P/VAX against cariogenic bacterial challenge. Groups of rats were orally challenged with S. mutans or Streptococcus sobrinus (S. sobrinus) and then immunized with pGJA-P/VAX or the vector pVAX1 intranasally. Serum IgG and salivary IgA antibody levels were assessed by an enzyme-linked immunosorbent assay and caries activity was evaluated by the Keyes method. The results showed that specific salivary IgA antibody responses were induced following intranasal vaccination with pGJA-P/VAX. Moreover, immunization with pGJA-P/VAX resulted in significantly reduced enamel and dentinal caries lesions in rats after S. mutans infection and significantly reduced enamel caries lesions after S. sobrinus infection. Thus, pGJA-P/VAX was not only protective toward S. mutans infection, but also provided cross-strain protection against S. sobrinus infection in rats.