The co-ordination and synchronization of DNA replication, chromosome partitioning and cell division in bacteria are critical to survival. In Escherichia coli, the septal protein FtsK links cell division and chromosome segregation through its integral membrane N-terminal and cytoplasmic C-terminal domains. FtsK is responsible for promoting decatenation and dimer resolution in the later stages of chromosome segregation by activating recombination at dif by the site-specific Xer recombinases. Here, we formally demonstrate, using novel assay based on real-time quantitative polymerase chain reaction, that dif recombination depends not only on proteins upstream of FtsK in the septum assembly pathway, but also on the activity of downstream proteins. Work in synchronized cell cultures further showed that even though FtsK is recruited early to the septum, dif recombination only occurs shortly before cell division and this activity requires a closing septum. We propose a model whereby septum localization and concentration of FtsK co-ordinate its various roles in chromosome segregation and cell division.