Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis, and detoxified PT is a crucial antigen of acellular pertussis vaccine. Here, plasmid DNA expressing the pertussis toxin S1 subunit (pcDNA/S1) of B. pertussis was evaluated for immunogenicity and for the ability to induce protection against PT challenge or B. pertussis infection in mice. The gene gun delivery of pcDNA/S1, performed by inserting the S1 gene into a mammalian expression vector, successfully induced anti-PT IgG antibody production. Immunization of mice with pcDNA/S1 significantly inhibited leukocytosis-promoting activity caused by PT or B. pertussis. In addition, pcDNA/S1 induced significant protection against intracerebral challenge with a lethal dose of B. pertussis. The results of the present study demonstrated that a DNA vaccine encoding the PT-S1 subunit induced protection against B. pertussis infection in mice. Thus, this vaccine preparation is potentially applicable for the production of novel vaccines against B. pertussis infection.