The small GTPase rho is functionally involved in the formation of cytoskeletal structures like stress fibers or focal adhesion plaques. Shigella entry into HeLa cells induces a blossom-like membrane structure at the bacterial entry site. We show here that this membrane-folding process is rho-dependent. The three rho isoforms were recruited into bacterial entry sites with differential localization relative to the membrane structure. A rho-specific inhibitor abolished Shigella-induced membrane folding and impaired bacterial entry accordingly. S1-myosin labeling indicated that rho was involved in Shigella-induced actin polymerization but not actin nucleation in the bacterial invasion site. This provides a major link in the signalization cascade allowing entry of a bacterial pathogen into a eukaryotic cell.