The cellular function of Escherichia coli topoisomerase III remains elusive. We show that rescue of temperature-sensitive mutants in parE and parC (encoding the subunits of the chromosomal decatenase topoisomerase IV) at restrictive temperatures by high-copy suppressors is strictly dependent on topB (encoding topoisomerase III). Double mutants of parEΔtopB and parCΔtopB were barely viable, grew slowly, and were defective in chromosome segregation at permissive temperatures. The topB mutant phenotype did not result from accumulation of toxic recombination intermediates, because it was not relieved by mutations in either recQ or recA. In addition, in an otherwise wild-type genetic background, ΔtopB cells treated with the type II topoisomerase inhibitor novobiocin displayed aberrant chromosome segregation. This novobiocin sensitivity was attributable to an increased demand for topoisomerase IV and is unlikely to define a new role for topoisomerase III; therefore, these results suggest that topoisomerase III participates in orderly and efficient chromosome segregation in E. coli.