The foodborne pathogen Listeria monocytogenes is able to colonize the human and animal intestinal tracts and subsequently crosses the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive and adapt to the low pH environment of the stomach. Gene sequence analysis indicates that lmo0043, an orthologue of arcA, encodes a protein containing conserved motifs and critical active amino acids characteristic of arginine deiminase that mediates an arginine deimination reaction. We attempted to characterize the role of ArcA in acid tolerance in vitro and in mice models. Transcription of arcA was significantly increased in L. monocytogenes culture subjected to acid stress at pH 4.8, as compared with that at pH 7.0. Deletion of arcA impaired growth of L. monocytogenes under mild acidic conditions at pH 5.5, and reduced its survival in synthetic human gastric fluid at pH 2.5 and in the murine stomach. Bacterial load in the spleen of mice intraperitoneally inoculated with an arcA deletion mutant was significantly lower than that of the wild-type strain. These phenotypic changes were recoverable by genetic complementation. Thus, we conclude that L. monocytogenes arcA not only mediates acid tolerance in vitro but also participates in gastric survival and virulence in mice.