Significance Pneumolysin, a pore-forming toxin of Streptococcus pneumoniae , assembles into rings on cholesterol-containing membranes of host cells. β -hairpins form a barrel-shaped transmembrane β -sheet that perforates the membrane, leading to cell lysis. In atomistic and coarse-grained molecular dynamics simulations, we resolve how pneumolysin docks to cholesterol-rich bilayers and how membrane pores form. Remarkably, pneumolysin rings are just wide enough that pores form even if lipid efflux is blocked, driven by spontaneous buckling of the lipid plug. In a survey of electron microscopy and atomic force microscopy images, we identify key intermediates along both the lipid-efflux and plug-buckling pathways of pore opening.