Pore-forming toxins (PFTs) expressed by bacteria oligomerize on target cells, causing cell lysis and promoting bacterial infections. Cytolysin A (ClyA), an α-PFT, is known to undergo one of the largest conformational changes during the transition from the monomer to the dodecameric pore complex. Although pore formation can occur in the absence of cholesterol, enhanced kinetics of oligomerization has been observed in membranes containing cholesterol. Despite several aspects of the pore-forming pathway of ClyA being understood, a complete molecular picture of the interplay between the different segments of the ClyA monomer during the transformation to the membrane inserted protomer remains elusive. In this study, we perform a combined experimental and all-atom molecular dynamics (MD) study of several point mutated ClyA monomers to unravel the role of the membrane inserted β-tongue and N-terminus motifs. Erythrocyte turbidity assays and vesicle leakage experiments reveal a complete loss of activity for β-tongue mutants, Y178F and A179F, and delayed activity for the N-terminus mutants, Y27A and Y27F. MD simulations reveal a distinct reduction in flexibility for the β-tongue residues for both Y178F and A179F monomers. This correlates positively with the loss of activity attributed to the compromised flipping out of crucial β-tongue residues during the monomer membrane binding step. For the N-termini mutants, delayed kinetics suggest retardation in the formation of oligomeric pore states and a reduced population of active pore states. In conclusion, the β-tongue is crucial for initiating the conformational change that precedes membrane binding and oligomerization for ClyA. In contrast, the N-terminus drives the transition from a pre-pore state to the membrane inserted pore state and maintains the integrity of the transmembrane channel. Our study suggests that conformational flexibility might have a direct bearing on our understanding of the complex problem of membrane induced protein folding and intermediate states.