The actin-like MreB cytoskeletal protein and RNA polymerase (RNAP) have both been suggested to provide the force for chromosome segregation. Here, we identify MreB and RNAP as in vivo interaction partners. The interaction was confirmed using in vitro purified components. We also present convincing evidence that MreB and RNAP are both required for chromosome segregation in Escherichia coli. MreB is required for origin and bulk DNA segregation, whereas RNAP is required for bulk DNA, terminus, and possibly also for origin segregation. Furthermore, flow cytometric analyses show that MreB depletion and inactivation of RNAP confer virtually identical and highly unusual chromosome segregation defects. Thus, our results raise the possibility that the MreB-RNAP interaction is functionally important for chromosome segregation.