Listeriolysin O (LLO) is a cholesterol-dependent pore-forming cytolysin secreted by the intracellular pathogen Listeria monocytogenes and a key mediator of bacterial virulence. LLO binds cholesterol-containing membranes and, under acidic conditions, assembles into pore-forming oligomers that disrupt the phagosomal membrane, enabling bacterial escape into the host-cell cytosol. As a member of the cholesterol-dependent cytolysin family, LLO serves as an important model for investigating membrane recognition, pore formation, pH-dependent toxin activity, and host–pathogen interactions. This recombinant protein is suitable for structural and functional studies, membrane-binding and pore-formation assays, antibody discovery and characterization, epitope mapping, and other toxin mechanism research applications.