Listeriolysin O (LLO) is the most crucial virulence factor of a food-borne pathogen Listeria monocytogenes. LLO is unique amongst non Listeria-derived cholesterol-dependent cytolysins, a family of pore forming toxins from Gram positive bacteria, in being stable and working optimally at low pH values. LLO allows phagosome-entrapped bacteria to escape to the cytoplasm by creating transmembrane pores in lipid membranes. The structural features of the pores are not yet completely understood, neither the dependence on lipid composition or pH. Here we used lipid membranes composed of 1,2-palmitoyl-sn-glycerophosphocholine and cholesterol at 1:1 (mol:mol) to study features of LLO pores. By using planar lipid bilayers we show that LLO is able to efficiently form pores at pH 5.5, as well as at physiological pH 7.4. Atomic force microscopy imaging revealed that pores were of the same size, however, the distribution of pore conductances was significantly different between the two pH values as observed by planar lipid membranes approach. Mutation of a LLO-unique residue H311 resulted in changed conformational stability of LLO monomer, rate of pore formation and conductivity of the pores. The results altogether indicate that β-barrel pores exhibit significant pH-dependent plasticity, not anticipated before.