Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti- diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtb PW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.