Mycobacterium bovis Bacille Calmette-Guérin (BCG) was developed as an attenuated live vaccine for tuberculosis control nearly a century ago. Despite being the most widely used vaccine in human history, the mechanisms of attenuation of BCG remain poorly understood. BCG is not a single organism, but comprises a number of substrains that differ in genotypes and phenotypes. The impacts of these differences on BCG vaccine properties are largely unknown. Nevertheless, in the past decade, the development of sophisticated genome analysis techniques, coupled with advances in knowledge of the virulence mechanisms of Mycobacterium tuberculosis, have provided greater insights into the attenuation and evolution of BCG. This review article discusses these new developments, focusing on molecular mechanisms that contribute to the attenuation of BCG substrains. It is evident that BCG strains comprise natural mutants of major virulence factors of M. tb, including ESX-1, PDIM/PGL and PhoP, and that BCG substrains differ markedly in virulence level. The impacts of these findings on vaccine properties including adverse reaction effect, tuberculin reactivity and protective efficacy are discussed. These new insights have extremely important implications for national immunization programs and the development of future vaccines.