Although the deletion of RD1 is likely correlated to attenuation from virulence for members of the Mycobacterium tuberculosis (MTB) complex, the reasons for this phenotype remain to be fully explained. As genomic variation is responsible for at least a component of variability in gene expression, we looked to the in vitro global expression profile of the RD1 artificial knockout from M. tuberculosis H37Rv (H37Rv:ΔRD1) for clues to elucidate its phenotypic shift towards attenuation. By comparing the transcriptome of H37Rv:ΔRD1 to that of virulent H37Rv, 15 regulated genes located in nine different regions outside of RD1 have been identified, capturing an effect of RD1's deletion on the rest of the genome. To assess whether these regulations are characteristic of attenuated MTB in general, expression profiles of natural RD1 mutants (BCG Russia, BCG Pasteur, and M. microti) as well as the ‘avirulent’ M. tuberculosis H37Ra, whose RD1 region is genomically intact, were obtained. Results indicate that attenuated strains lack the expression of RD1 genes including cfp10 and esat6, whether through deletion or reduced expression. Furthermore, comparative transcriptomics reveals the concurrent down-regulation of several gene neighborhoods beyond RD1. The potential relevance of these other expression changes towards MTB virulence is discussed.