The capacity of Listeria monocytogenes to withstand low pH is important for growth in low-pH foods, successful passage through the gastric barrier and survival within the macrophage phagosome. The ability of this pathogen to survive and adapt to acidic conditions is therefore predicted to play a significant role in the infectious cycle. In silico analysis of the L. monocytogenes genome revealed the presence of putative arginine deiminase (ADI) genes, which have been shown to play a role in the acid tolerance of other bacterial genera. In the present study, we show that L. monocytogenes possesses a functional ADI system and analysis of deletion mutants reveals that it contributes to both growth and survival of the bacterium under acidic conditions. An RT-PCR approach demonstrated that expression of ADI genes is increased in environments of low pH and anaerobicity and in the presence of arginine. A putative activator of ADI genes, namely ArgR, was identified and was shown to contribute to transcriptional regulation at this locus. Furthermore, expression of ADI genes was shown to be modulated by both the alternative stress sigma factor σB and the central virulence regulator PrfA. Finally, using the murine model of infection, we have established a role for the ADI system in the virulence of L. monocytogenes.