Pore formation is central to the ability of cholesterol dependent cytolysins (CDCs) to act as important bacterial virulence factors. Secreted by Gram-positive pathogens, the toxins self-assemble on the target membrane to form large circular proteinaceous pores 200-300 Å in diameter. Following the current model of CDC pore formation monomers first bind the target membrane via Domain 4 and then self-assemble into a circular ring termed the prepore. The subsequent vertical collapse of Domains 1/3 towards the membrane is associated with the deployment of a novel β-barrel architecture to achieve pore formation. It is also suggested that Domain 2, a twisted sheet connecting Domain 4 and Domains 1/3 experiences a substantial structural change. However, this conformational rearrangement remains uncharacterized. We investigate here this issue employing molecular modelling and structural bioinformatics. Systematic analysis of monomeric CDC structures allows us to define the conformational properties of Domain 2. This work shows that concerted rotational movement of Domain 2 throughout the oligomeric assembly best describes the prepore-to-pore structural transition. Further support for this mechanism comes from analysis of available cryo-electron microscopy maps. The work presented here procures the most plausible and testable structural mechanism of CDC oligomeric transition to achieve pore formation.