In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies. These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures. We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins. Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L. monocytogenes and had no effect on the actin-based motility of this bacterial parasite. Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.