In Escherichia coli, the replication initiator DnaA oscillates between an ATP- and an ADP-bound state in a cell cycle-dependent manner, supporting regulation for chromosome replication. ATP–DnaA cooperatively assembles on the replication origin using clusters of low-affinity DnaA-binding sites. After initiation, DnaA-bound ATP is hydrolyzed, producing initiation-inactive ADP–DnaA. For the next round of initiation, ADP–DnaA binds to the chromosomal locus DARS2, which promotes the release of ADP, yielding the apo-DnaA to regain the initiation activity through ATP binding. This DnaA reactivation by DARS2 depends on site-specific binding of IHF (integration host factor) and Fis proteins and IHF binding to DARS2 occurs specifically during pre-initiation. Here, we reveal that Fis binds to an essential region in DARS2 specifically during pre-initiation. Further analyses demonstrate that ATP–DnaA, but not ADP–DnaA, oligomerizes on a cluster of low-affinity DnaA-binding sites overlapping the Fis-binding region, which competitively inhibits Fis binding and hence the DARS2 activity. DiaA (DnaA initiator-associating protein) stimulating ATP–DnaA assembly enhances the dissociation of Fis. These observations lead to a negative feedback model where the activity of DARS2 is repressed around the time of initiation by the elevated ATP–DnaA level and is stimulated following initiation when the ATP–DnaA level is reduced.