Gram-positive bacteria, including the common wound-infecting Staphylococcus aureus, the virulent food-borne pathogen Listeria monocytogenes, and Streptococcus pneumoniae that causes pneumonia, utilize cholesterol-dependent cytolysins (CDCs) to attack and kill mammalian and human cells (Gilbert, 2005; Tweten, 2005). The bacteria produce and release CDCs as water-soluble monomers that attach to cholesterol-containing cell membranes, where they assemble into large, 200–500 Å cytolytic pores (Olofsson et al., 1993). X-ray structures of pneumolysin (PLY) from S. pneumoniae (Lawrence et al., 2015; Marshall et al., 2015; van Pee et al., 2016) show that the soluble toxin monomers are roughly rod-shaped and consist of four domains (D1-D4). Sequence comparison suggests that all CDCs have the same domain structure and therefore insert into target membranes in essentially the same way (Tweten et al., 2001). Although numerous biochemical studies have addressed the arrangement of monomers in the CDC prepore or pore complex (Hotze and Tweten, 2012; Tilley et al., 2005) and how they might penetrate the lipid bilayer (Shatursky et al., 1999), the detailed structure of the pore complex, and hence the exact mechanism of membrane insertion is unknown. The size heterogeneity of CDC prepores and pores has so far precluded structure determination at high resolution. We determined the structure of the ring-shaped ~2.2 MDa PLY pore complex by cryoEM. Together with our 2.4 Å x-ray structure of soluble PLY (van Pee et al., 2016) and a map of the prepore complex obtained by electron cryo-tomography, we can now describe the entire process of membrane attachment, prepore and pore formation in near-atomic detail. Given the high degree of sequence conservation amongst CDCs, it is likely that the same mechanism of membrane insertion holds for related bacterial toxins.