Background: Many bacteria transport cellobiose via a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). In Listeria monocytogenes, two pairs of soluble PTS components (EIIA<sup>Cel1</sup>/EIIB<sup>Cel1</sup> and EIIA<sup>Cel2</sup>/EIIB<sup>Cel2</sup>) and the permease EIIC<sup>Cel1</sup> were suggested to contribute to cellobiose uptake. Interestingly, utilization of several carbohydrates, including cellobiose, strongly represses virulence gene expression by inhibiting PrfA, the virulence gene activator. Results: The LevR-like transcription regulator CelR activates expression of the cellobiose-induced PTS operons celB1-celC1-celA1, celB2-celA2, and the EIIC-encoding monocistronic celC2. Phosphorylation by P∼His-HPr at His550 activates CelR, whereas phosphorylation by P∼EIIB<sup>Cel1</sup> or P∼EIIB<sup>Cel2</sup> at His823 inhibits it. Replacement of His823 with Ala or deletion of both celA or celB genes caused constitutive CelR regulon expression. Mutants lacking EIIC<sup>Cel1</sup>, CelR or both EIIA<sup>Cel</sup> exhibitedslow cellobiose consumption. Deletion of celC1 or celR prevented virulence gene repression by the disaccharide, but not by glucose and fructose. Surprisingly, deletion of both celA genes caused virulence gene repression even during growth on non-repressing carbohydrates. No cellobiose-related phenotype was found for the celC2 mutant. Conclusion: The two EIIA/B<sup>Cel</sup> pairs are similarly efficient as phosphoryl donors in EIIC<sup>Cel1</sup>-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the celA double mutant further supports a role of PTS<sup>Cel</sup> components in PrfA regulation.