Pathogenic bacteria of the genus Yersinia counteract host defense by interfering with eukaryotic signal transduction pathways. YpkA of Yersinia pseudotuberculosis shares significant homology with eukaryotic Ser/Thr protein kinases, is translocated into the host cell and has been shown to be an essential virulence factor in a mouse infection model. In this study, we identify the small GTPases RhoA and Rac-1 as eukaryotic binding partners of YpkA and its homolog YopO of Yersinia enterocolitica. We demonstrate that the interaction is independent of phosphorylation of YpkA and nucleotide loading state of the GTPases. The interaction with RhoA and Rac-1 might provide an important clue to how YpkA interferes with eukaryotic signaling on a molecular level.