An ability to translocate the mucosal epithelia through M cells provides invasive pathogens with a rapid means of accessing the mucosal lymphoid tissues. In order to determine the role of M cells in Listeria monocytogenes infection, we initially assessed colonization of Peyer's patch (PP) epithelium in BALB/c mice by Vibrio cholerae Eltor, wild-type L. monocytogenes and an isogenic hemolysin mutant (LO28Deltahly). It was observed that both wild-type L. monocytogenes and Deltahly showed preferential colonization of PP epithelium in this model. Furthermore, a novel luciferase reporter system was used to show rapid site-specific localization of L. monocytogenes in intestinal Peyer's patches. To examine the role of M cells in transcytosis of L. monocytogenes we utilized an in vitro transwell model that mimics M-cell activity through differentiation of C2Bbe1 epithelial enterocytes via co-culture with murine Peyer's patch lymphocytes (PPL). It was shown that L. monocytogenes transits M cells at significantly increased rates compared to C2Bbe1 monocultures. In addition, M-cell transport occurred independently of bacterial hemolysin and internalin production. This study demonstrates rapid transcytosis of L. monocytogenes through M cells, a process that occurs independently of the action of classical virulence factors.