The BCG vaccine (Bacille Calmette-Guerin), only prophylactic measure against tuberculosis (TB), was obtained in the early twentieth century by Calmette and Guérin after 231 passages of a M. bovis clinical isolate in medium containing glycerin and bovine bile. Its protective efficacy against pulmonary TB in adults varies from 0-80% and the genetic differences among vaccines strains used worldwide contribute to this variation. The Brazilian vaccine strain, BCG Moreau, is considered a primitive strain and more immunogenic, closer to the original BCG when compared to newer strains, such as BCG Pasteur (reference strain). The characterization of BCG sub-strains can contribute not only to a better understanding of the vaccine and its protective effect, but also, in elucidating how different BCG culture conditions may contribute to the impact on the host's immune response. Thus, we aimed to characterize the differences in gene expression through the intracellular proteomic profile of BCG Moreau and Pasteur strains, cultivated in Sauton or 7H9 medium, using the methodology of two-dimensional electrophoresis (2DE) and mass spectrometry.