SummaryQuantitative RT‐PCR revealed that transcripts of all four putative DEAD‐box RNA helicase genes of the psychrotrophic pathogen Listeria monocytogenes EGD‐e are found at higher levels in organisms grown at 3°C than at 37°C. At 3°C, growth of the three corresponding gene deletion mutants Δlmo0866, Δlmo1450 and Δlmo1722 was clearly restricted. The minimum growth temperatures of the three mutants were also higher than that of the wild‐type EGD‐e. In addition to inability to grow at 3°C, growth of Δlmo0866 and Δlmo1722 was reduced at 25°C, suggesting special roles of Lmo0866 and Lmo1722 in growth at suboptimal temperatures. Growth of Δlmo1450 was restricted not only at 3°C and 25°C, but also at 37°C, suggesting that Lmo1450 plays a universal role in growth of L. monocytogenes EGD‐e. Moreover, cold‐sensitive Δlmo0866, Δlmo1450 and Δlmo1722 were impaired in motility. The Δlmo0866 and Δlmo1450 strains were non‐motile, while Δlmo1722 showed reduced motility. This study shows that the putative DEAD‐box RNA helicase genes lmo0866, lmo1450 and lmo1722 are necessary for cold tolerance and motility of L. monocytogenes EGD‐e.