Bordetella pertussis, the etiologic agent of whooping cough, is a highly infectious pathogen with a strong capacity to colonize the human respiratory tract. A single infection with virulent B. pertussis induces strong mucosal and systemic humoral and cellular immune responses, as well as long-lasting protection in humans. Therefore, B. pertussis may be a suitable vector for intranasal vaccination against a variety of diseases by a single dose. We showed that intranasally administered recombinant live B. pertussis expressing heterologous antigens induce mucosal immune responses in the respiratory and the genital tracts of mice as well as systemic antibody responses. To consider live B. pertussis for human vaccination, we, therefore, aimed at the development of attenuated strain. Allelic exchange was used to delete the dermonecrotic toxin gene and to replace the pertussis toxin gene by an allele encoding inactive toxin. To reduce the production of tracheal cytotoxin, the ampG gene was over-expressed. After various rounds of homologous recombination, B. pertussis BPZE, affected in the production or activity of three major toxins, was isolated. Histological examinations of infected mice confirmed the strong attenuation of this strain. Nevertheless, it colonized the mouse respiratory tract over a period of roughly 1 month. The level of protection against subsequent challenge with virulent wild-type B. pertussis, induced by a single intranasal administration of BPZE was at least as high as that induced by two injections of commercial acellular vaccine. We conclude that live attenuated B. pertussis may be an attractive vaccine candidate to be administered in a single intranasal dose against whooping cough. Moreover, live recombinant B. pertussis may be used as a vector for intranasal vaccination against a variety of diseases.