Protein erylysin A (EryA) belongs to the protein family of aegerolysins (Pfam 06355; InterPro IPR009413) and is produced by the king oyster mushroom (Pleurotus eryngii). EryA specifically binds to the main invertebrate sphingolipid, ceramide phosphoethanolamine. Point mutations were introduced by mutagenesis to replace selected amino acid residues with alanine (protein variants EryA(W28A), EryA(E69A), EryA(T98A), EryA(W96A), EryA(K99A)) and one with tyrosine (protein variant EryA(A91Y)). These amino acid residues were selected on the basis of previous studies, on the binding of variants of aegerolysins ostreolysin A6 (OlyA6) and pleurotolysin A2 (PlyA2) to sphingomyelin, when combined with cholesterol, and their high degree of similarity with EryA. From the alignment visualization of structure OlyA6 with structure model EryA, we concluded that the protein binding pockets are most likely very similar. EryA does not have the preserved tyrosine residue Y91, which is present in OlyA6 and PlyA2 proteins. Together with tryptophan at site 28 and 96, Y91 is thought to form a lipid binding site. Unlike OlyA6 and PlyA2, EryA does not bind to sphingomylein/cholesterol complexes. Therefore, we concluded that the answer for specific EryA interaction with ceramide phosphoethanolamine could lie here. We successfully isolated the recombinant EryA protein and five variants thereof. We demonstrated that the variants EryA(A91Y) and EryA(T98A) have comparable affinity for binding to ceramide phosphoethanolamine, when combined with cholesterol, as EryA. Consequently, the EryA (A91Y)/PlyB and EryA(T98A)/PlyB complexes have the same cytolytic activity as the EryA/PlyB complex. At the same time, we have shown that amino acid residue of alanine at position 91 is not crucial for the specific binding of the EryA to ceramide phosphoethanolamine.