Listeriolysin O (LLO) is a thiol-activated cytolysin secreted by Listeria monocytogenes. LLO and phosphatidylinositol phospholipase C are two essential virulence factors, which this bacterium needs to escape from the phagosomal compartment to the cytoplasm. Cathepsin-D specifically cleaves LLO, between the Trp-491 (tryptophan amino acid in three letter nomenclature) and Trp-492 residues of the conserved undecapeptide sequence, ECTGLAWEWWR, in the domain 4 of LLO (D4). Moreover, these residues also correspond to the phagosomal-binding epitope. Cathepsin-D had no effect on phosphatidylinositol phospholipase C. We have observed that cathepsin-D cleaved the related cholesterol-dependent cytolysin pneumolysin at the same undecapeptide sequence between Trp-435 and Trp-436 residues. These studies also revealed an additional cathepsin-D cleavage site in the pneumolysin D4 domain localized in the 361-GDLLLD-366 sequence. These differences might confer a pathogenic advantage to listeriolysin O, increasing its resistance to phagosomal cathepsin-D action by reducing the number of cleavages sites in the D4 domain. Using ΔLLO/W491A and ΔLLO/W492A bacterial mutants, we reveal that the Trp-491 residue has an important role linked to cathepsin-D in Listeria innate immunity.