While different features for the activity of the bacterial canonical SMC complex, Smc-ScpAB, have been described in different bacteria, not much is known about the way chromosomes in enterobacteria interact with their SMC complex, MukBEF. Here we used a number of in vivo assays in E. coli to reveal how MukBEF controls chromosome conformation and how the MatP/ matS system prevents MukBEF activity. Our results indicate that the loading of MukBEF occurs preferentially in newly replicated DNA, at multiple loci on the chromosome where it can promote long-range contacts in cis even though MukBEF can promote long-range contacts in the absence of replication. Using HiC and ChIP-seq analyses in strains with rearranged chromosomes, the prevention of MukBEF activity increases with the number of matS sites and this effect likely results from the unloading of MukBEF by MatP. Altogether, our results reveal how MukBEF operates to control chromosome folding and segregation in E. coli.