The gram-positive bacterium Listeria monocytogenes is a food-borne pathogen of both public health and food safety significance. It possesses three small, highly homologous protein members of the cold shock protein (Csp) protein family. We used gene expression analysis and a set of mutants with single, double and triple deletion of the csp genes to evaluate roles of CspA, CspB and CspD in cold and osmotic (NaCl) stress adaptation responses of L. monocytogenes. All three Csps are dispensable for growth at optimal temperature (37°C). These proteins are, however, required for efficient cold and osmotic stress tolerance of this bacterium. The hierarchy of their functional importance differs depending on the environmental stress conditions: CspA>CspD>CspB in response to cold stress versus CspD>CspA/CspB in response to NaCl salt osmotic stress. The fact that Csps are promoting L. monocytogenes adaptation against both cold and NaCl stress has significant implications in view of practical food microbial control measures. The combined or sequential exposure of L. monocytogenes cells to these two stresses in food environments might inadvertently induce cross protection responses.