The problems of tuberculosis (TB) and its drug resistance are very severe in China. New therapeutic agents or regimens to treat multi-drug-resistant tuberculosis (MDR-TB) are urgently needed. In this study, the effects of Ag85A DNA or ESAT6/Ag85A chimeric DNA vaccines alone or in combination with rifampin (RFP) were studied for the treatment of mice with MDR-TB. Eighty female BALB/c mice infected with Mycobacterium tuberculosis clinical isolate HB361, which was resistant to high level of RFP, and low level of isoniazid (INH), were treated with the saline, plasmid vector pVAX1, RFP, HSP65 DNA, Ag85A DNA, Ag85A DNA combined with RFP, chimeric ESAT6/Ag85A DNA, chimeric ESAT6/Ag85A DNA combined with RFP, respectively. Different effects of DNA vaccines for the treatment of MDR-TB were demonstrated in this study. Compared with saline group, Ag85A DNA vaccine alone or Ag85A DNA in combination with rifampin group reduced the pulmonary and splenic bacterial loads by 0.58, 0.82 and 0.51, 0.69 logs, respectively. The pathological changes of lungs were also slight and the lesions were limited in comparison with that of the control mice in which the lesions were extensive and more necrotic changes were observed. Interestingly, the chimeric Ag85A/ESAT6 DNA vaccine showed the lower effect for the treatment of MDR-TB. Ag85A DNA vaccine played a main role for the treatment of TB and MDR-TB. We believe that this is the first report of the use of DNA vaccine in the treatment of MDR-TB, and that these data suggest that DNA vaccine was effective for the treatment of MDR-TB which might have the potential contribution for resolving this problem in developing countries.