Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming proteins making up a major class of virulence factors. They are secreted as soluble monomers that bind eukaryotic membranes and form oligomeric rings before undergoing a series of irreversible structural rearrangements leading to pore formation. While CDCs target host membranes in different ways, they all require cholesterol to pierce the lipid bilayer. We have used electron cryo-microscopy (cryoEM) to solve the structure of the CDC intermedilysin trapped as an early prepore on lipid nanodiscs. The cryoEM map highlights the movement of a key amphipathic helix in the earlier steps of pore formation, which is positioned parallel to the membrane. Liposome lysis assays and mutagenesis show that this helix is a determinant of lytic activity: by changing the charge composition of this horizontal helix, we were able to tune the activity of CDCs and even overcome the need for cholesterol. Our results suggest that the horizontal helix disrupts the membrane with its charged residues before rupturing it. Equivalent amphipathic membrane-interacting helices are found in other membrane-lysing proteins such as the eukaryotic protein gasdermin and antimicrobial peptides. Together, our results provide insight into the mechanism of protein-mediated membrane lysis and possible ways of engineering CDCs to target specific lipids.