Streptolysin S (SLS)-like virulence factors from clinically relevant Gram-positive pathogens have been proposed to behave as potent cytotoxins, playing key roles in tissue infection. Listeriolysin S (LLS) is an SLS-like hemolysin/bacteriocin present amongListeria monocytogenesstrains responsible for human listeriosis outbreaks. As LLS cytotoxic activity has been associated with virulence, we investigated the LLS-specific contribution to host tissue infection. Surprisingly, we first show that LLS causes only weak red blood cell (RBC) hemolysisin vitroand neither confers resistance to phagocytic killing nor favors survival ofL. monocytogeneswithin the blood cells or in the extracellular space (in the plasma). We reveal that LLS does not elicit specific immune responses, is not cytotoxic for eukaryotic cells, and does not impact cell infection byL. monocytogenes. Usingin vitrocell infection systems and a murine intravenous infection model, we actually demonstrate that LLS expression is undetectable during infection of cells and murine inner organs. Importantly, upon intravenous animal inoculation,L. monocytogenesis found in the gastrointestinal system, and only in this environment LLS expression is detectedin vivo. Finally, we confirm that LLS production is associated with destruction of target bacteria. Our results demonstrate therefore that LLS does not contribute toL. monocytogenestissue injury and virulence in inner host organs as previously reported. Moreover, we describe that LlsB, a putative posttranslational modification enzyme encoded in the LLS operon, is necessary for murine inner organ colonization. Overall, we demonstrate that LLS is the first SLS-like virulence factor targeting exclusively prokaryotic cells duringin vivoinfections.IMPORTANCEThe most severe human listeriosis outbreaks are caused byL. monocytogenesstrains harboring listeriolysin S (LLS), previously described as a cytotoxin that plays a critical role in host inner tissue infection. Cytotoxic activities have been proposed as a general mode of action for streptolysin S (SLS)-like toxins, including clostridiolysin S and LLS. We now challenge this dogma by demonstrating that LLS does not contribute to virulencein vivoonce the intestinal barrier has been crossed. Importantly, we show that intravenousL. monocytogenesinoculation leads to bacterial translocation to the gastrointestinal system, where LLS is specifically expressed, targeting the host gut microbiota. Our study highlights the heterogeneous modes of action of SLS-like toxins, and we demonstrate for the first time a further level of complexity for SLS-like biosynthetic clusters as we reveal that the putative posttranslational modification enzyme LlsB is actually required for inner organ colonization, independently of the LLS activity.