Cholesterol dependent cytolysins (CDCs) are bacterial pore-forming toxins which are secreted and directed towards cholesterol-rich host cell membranes. Upon targeting specific membrane components, they oligomerize into complete ring or arc-like structures and perforate the membrane bilayer. Even though the key membrane component for CDCs to oligomerize is cholesterol, in some cases other membrane associated molecules were shown to assist it. Vaginolysin (VLY) from Gardnerella vaginalis belongs to a small group of CDCs dependent on CD59, a human cell marker. In this study we investigated the role of CD59 during VLY pore formation. Using analytical ultracentrifugation, we showed that VLY stays in a monomeric state prior to encountering the membrane. Using negative staining electron microscopy, we found that CD59 alone can trigger VLY oligomerization, but this happens only when the receptor is attached to the membrane. However, CD59-driven VLY oligomerization does not lead to functional pores – a cholesterol-rich membrane is irreplaceable here. Moreover, we managed to capture the difference in the kinetics of pore formation on cholesterol-rich liposomes with and without CD59, and as CD59 is placed further away from the membrane surface, using a sulforhodamine B-encapsulated liposome leakage assay. CD59 attached to the cholesterol-rich liposomes enhances the rate of liposome leakage. Our findings suggest that the role CD59 plays in VLY pore formation is to expedite oligomerization upon interaction with the membrane. This may happen via VLY orientation and alignment at the membrane interface or by shifting the mechanism towards arc-shaped pores.