The tubulin-like FtsZ protein initiates assembly of the bacterial cytokinetic machinery by polymerizing into a ring structure, the Z ring, at the prospective site of division. To block Z-ring formation over the nucleoid and help coordinate cell division with chromosome segregation, Escherichia coli employs the nucleoid-associated division inhibitor, SlmA. Here, we investigate the mechanism by which SlmA regulates FtsZ assembly. We show that SlmA disassembles FtsZ polymers in vitro. In addition, using chromatin immunoprecipitation (ChIP), we identified 24 SlmA-binding sequences (SBSs) on the chromosome. Remarkably, SlmA binding to SBSs dramatically enhanced its ability to interfere with FtsZ polymerization, and ChIP studies indicate that SlmA regulates FtsZ assembly at these sites in vivo. Because of the dynamic and highly organized nature of the chromosome, coupling SlmA activation to specific DNA binding provides a mechanism for the precise spatiotemporal control of its anti-FtsZ activity within the cell.