The low-virulence Listeria monocytogenes strains have been previously assigned to 4 phenotypic groups. This study aimed to characterize the A23 strain, which exhibits a pulsed-field gel electrophoresis profile specific to low-virulence strains. This strain has the same causal mutations as the group III strains and a supplementary mutation in the mpl gene, leading to the absence of internalin A expression and the presence of inactive internalin B, phosphatidyl-inositol phospholipase C, and phosphatidylcholine phospholipase C. Despite these mutations in major virulence genes, the A23 strain formed plaques in cell monolayers and contaminated 100% of inoculated mice, suggesting that it evolved from group III strains by acquiring new virulence genes.