Listeria monocytogenes is an environmental bacterium that becomes a pathogen following ingestion by a mammalian host. The transition from environmental organism to pathogen requires significant changes in gene expression, including the increased expression of gene products that contribute to bacterial growth within host cells. PrfA is an L. monocytogenes transcriptional regulator that becomes activated upon bacterial entry into mammalian cells and induces the expression of gene products required for virulence. How PrfA activation occurs is not known, however several mutations have been identified that increase PrfA activity in strains grown in vitro (prfA mutations). Here we describe a novel prfA mutation that enhances extracellular PrfA-dependent gene expression but in contrast to prfA mutants inhibits the cytosol-mediated induction of virulence genes. prfA Y154C strains entered cells and escaped from phagosomes with an efficiency similar to wild type bacteria, however the mutation prevented efficient L. monocytogenes actin polymerization and reduced spread of bacteria to adjacent cells. The prfA Y154C mutation severely attenuated bacterial virulence in mice but the mutant strains did generate target antigen specific CD8(+) effector cells. Interestingly, the prfA Y154C mutant was significantly less cytotoxic for host cells than wild type L. monocytogenes. The prfA Y154C mutant strain may therefore represent a novel attenuated strain of L. monocytogenes for antigen delivery with reduced host cell toxicity.