By comparative genomics, we have identified a gene of the intracellular pathogen Listeria monocytogenes that encodes an LPXTG surface protein absent from nonpathogenic Listeria species. This gene, vip, is positively regulated by PrfA, the transcriptional activator of the major Listeria virulence factors. Vip is anchored to the Listeria cell wall by sortase A and is required for entry into some mammalian cells. Using a ligand overlay approach, we identified a cellular receptor for Vip, the endoplasmic reticulum (ER) resident chaperone Gp96 recently shown to interact with TLRs. The Vip-Gp96 interaction is critical for bacterial entry into some cells. Comparative infection studies using oral and intravenous inoculation of nontransgenic and transgenic mice expressing human E-cadherin demonstrated a role for Vip in Listeria virulence, not only at the intestine level but also in late stages of the infectious process. Vip thus appears as a new virulence factor exploiting Gp96 as a receptor for cell invasion and/or signalling events that may interfere with the host immune response in the course of the infection.