Survival of the food-borne pathogen Listeria monocytogenes in environments of elevated osmolarity and reduced temperature is attributed, at least in part, to the accumulation of the trimethylammonium compound glycine betaine. Previously we identified betL, a gene encoding the secondary glycine betaine transporter BetL, which we linked to the salt tolerance of Listeria. In this report, we demonstrate that betL, preceded by a consensus sigmaB-dependent promoter, is regulated by osmotic up-shock, at least in part at the level of transcription. Using allelic exchange mutagenesis we constructed an in-frame deletion in betL, and used this mutant to determine the role of BetL in contributing to the growth and survival of L. monocytogenes, both in a high risk food (Camembert cheese) and animal model. Our results indicate that while BetL plays an important role in glycine betaine mediated osmoprotection, mutating the gene does not significantly effect either the cryotolerance or virulence of the organism.