The replication of a bacterial chromosome is initiated by the DnaA protein, which binds to the specific chromosomal regionoriCand unwinds duplex DNA within the DNA-unwinding element (DUE). The initiation is tightly regulated by many factors, which control either DnaA ororiCactivity and ensure that the chromosome is duplicated only when the conditions favor the survival of daughter cells. The factors controllingoriCactivity often belong to the protein families of two-component systems. Here, we found thatHelicobacter pylori oriCactivity is controlled by HP1021, a member of the atypical response regulator family. HP1021 protein specifically interacts withH. pylori oriCat HP1021 boxes (5′-TGTT[TA]C[TA]-3′), which overlap with three modules important fororiCfunction: DnaA boxes, the hypersensitivity (hs) region and the DUE. Consequently, HP1021 binding tooriCprecludes DnaA-oriCinteractions and inhibits DNA unwinding at the DUE. Thus, HP1021 constitutes a negative regulator of theH. pyloriorisome assemblyin vitro. Furthermore, HP1021 boxes were found upstream of at least 70 genes, including those encoding CagA and Fur proteins. We postulate that HP1021 might coordinate chromosome replication, and thus bacterial growth, with other cellular processes and conditions in the human stomach.