Perfringolysin O (PFO) is a toxic protein that binds to cholesterol-containing membranes, oligomerizes and forms a β-barrel transmembrane pore, leading to cell lysis. Previous studies have uncovered the sequence of events in this multistage structural transition to a considerable detail, but the underlying molecular mechanisms are not yet fully understood. By measuring hydrogen-deuterium exchange patterns of peptide bond amide protons monitored by mass spectrometry (HDX-MS), we have mapped structural changes in PFO and its variant bearing a point mutation, during incorporation to the lipid environment. We have defined all regions which undergo structural changes caused by the interaction with the lipid environment, both in wild-type PFO, thus providing new experimental constraints for molecular modeling of the pore formation process, and also in a point mutant W165T, for which the pore formation process is known to be inefficient. We have demonstrated that a point mutation W165T causes destabilization of protein solution structure, strongest for domain D1, which interrupts the pathway of structural transitions in other domains necessary for proper oligomerization in the membrane. In PFO the strongest changes accompanying binding to the membrane focus in D1, D4 C-terminal part and strands β1, β4, β5 of D3. These changes were much weaker for PFOW165Tlipo, where substantial stabilization was observed only in D4 domain. In this study, the application of HDX-MS provided a new insight into conformational changes of PFO associated with the membrane binding, oligomerization, and lytic pore formation.