In Listeria monocytogenes EGDe, the lmo0501 gene locus encodes a protein similar to the mannitol transcription regulator (MltR) protein in Bacillus subtilis and Bacillus stearothermophilus. In this study we investigated its functional role in L. monocytogenes EGDe cells in view of growth under different stress conditions. Increased lmo0501 gene expression at mRNA level was detected in response to cold, osmotic and organic acid stress exposure. An EGDe Δlmo0501 mutant strain was diminished in growth compared to the wild type strain in minimal defined medium containing either glucose or fructose, as carbon sources. Growth of the lmo0501 null mutant was retarded growth under cold (4 °C), salt (NaCl) and organic acid stress conditions relative to the parental wild type strain. Our results confirm the role of the lmo0501 gene in adaptation of L. monocytogenes cells to food preservation stress conditions as well as to the efficient utilization of glucose and fructose as carbon sources.