Flagellar motility is an important process for bacterial survival. It allows bacteria to move to aquire nutrients or to retreat from unfavorable environments. In the Gram-positive bacterium Listeria monocytogenes, flagellar motility is a temperature-dependent process, being restricted to temperatures below 37°C. This regulation is mediated by the activities of the DegU response regulator, the MogR transcriptional repressor and GmaR, the MogR antirepressor. Transcriptional regulation of the fliN-gmaR promoter is central to flagellar motility regulation as pfliN-gmaR is transcriptionally activated by DegU and is also MogR repressed. However, the mechanism by which DegU mediates transcriptional activation of the fliN-gmaR promoter is not well understood. In this study, we further investigated the mechanisms controlling transcription initiating at the fliN-gmaR promoter by performing two comprehensive transposon mutagenesis screens to identify any additional factors involved in the activation and repression of pfliN-gmaR. The mutagenesis screen performed at 30°C to identify factors involved in pfliN-gmaR transcriptional activation led to the identification of a novel factor. We demonstrated that Lmo0866, a protein from the DEAD-box protein family, is required for transcriptional activation of pfliN-gmaR and therefore flagellar motility. Moreover, we identified CodY as being involved in the repression of pfliN-gmaR at 37°C.