Whooping cough is a highly contagious and acute respiratory disease which mainly affects children. Bordetella pertussis is responsible for a majority of these cases. Despite widespread vaccination, re-emergence of pertussis has been reported both in developed and developing countries. It is necessary to develop new vaccine candidates with high effectiveness. In this study, we constructed a live attenuated strain named BPTM1, in which two genes encoding major toxins PTX and DNT were removed, and a third gene, ampG, responsible for the transportation of toxin TCT was replaced by a homologous gene from Escherichia coli, by homologous recombination. The decreased expression of S1 of PTX was supported by Western bolting. The knock-out or replacement of three genes was confirmed at DNA level by polymerase chain reaction (PCR). The attenuated strain, BPTM1, showed growth curves in vitro and colonization curves in vivo similar to the parent strain BPMM. Although BPTM1 had a significant reduction in lung pathology than BMMM, it induced the same levels of antibodies IgG, IgG1, IgG2a as BPMM. These results suggest that attenuated Bordetella pertussis BPTM1 may become a candidate vaccine for pertussis vaccine.