Aerolysin is the founding member of a major class of β-pore-forming toxins (β-PFTs) found throughout all kingdoms of life. PFTs are cytotoxic proteins produced as soluble monomers, which oligomerize at the membrane of target host cells forming pores that may lead to osmotic lysis and cell death. Besides their role in microbial infection, they have become interesting for their potential as biotechnological sensors and delivery systems. Using an approach that integrates bioinformatics with molecular modeling and simulation, we looked for conserved features across this large toxin family. The cell surface-binding domains present high variability within the family to provide membrane receptor specificity. On the contrary, the novel concentric double β-barrel structure found in aerolysin is highly conserved in terms of sequence, structure and conformational dynamics, which likely contribute to preserve a common transition mechanism from the prepore to the mature pore within the family.Our results point to the key role of several amino acids in the conformational changes needed for oligomerization and further pore formation, such as Y221, W227, P248, Q263 and L277, which we propose are involved in the release of the stem loop and the two adjacent β-strands to form the transmembrane β-barrel.