AbstractPneumolysin is a cholesterol-dependent cytolysin (CDC) and virulence factor of Streptococcus pneumoniae. It kills cells by forming pores assembled from oligomeric rings in cholesterol-containing membranes. Cryo-EM has revealed the structures of the membrane-surface bound pre-pore and inserted-pore oligomers, however the molecular contacts that mediate these oligomers are unknown because high-resolution information is not available. Here we have determined the crystal structure of full-length pneumolysin at 1.98 Å resolution. In the structure, crystal contacts demonstrate the likely interactions that enable polymerisation on the cell membrane and the molecular packing of the pre-pore complex. The hemolytic activity is abrogated in mutants that disrupt these intermolecular contacts, highlighting their importance during pore formation. An additional crystal structure of the membrane-binding domain alone suggests that changes in the conformation of a tryptophan rich-loop at the base of the toxin promote monomer-monomer interactions upon membrane binding by creating new contacts. Notably, residues at the interface are conserved in other members of the CDC family, suggesting a common mechanism for pore and pre-pore assembly.