Attachment to mucosal surfaces is the initial event in the pathogenesis of the human foodborne pathogen Listeria monocytogenes. By use of comparative genomics, we identified a L. monocytogenes-specific gene, lapB, that encodes an LPXTG surface protein that is absent from nonpathogenic Listeria species. We showed that lapB expression is positively regulated by PrfA, the major transcriptional activator of the virulence genes of Listeria species, and is up-regulated in mouse spleens during infection. We demonstrated that LapB is an SrtA-anchored surface protein required for adhesion to and entry into mammalian cells and for virulence following intravenous or oral inoculation in mice. Our results highlight LapB as a new L. monocytogenes virulence adhesin with a function that is supported by its unique N-terminal domain through the probable interaction with a cellular receptor.