Bacteria regulate the frequency and timing of DNA replication initiation by controlling the activity of the replication initiator protein DnaA. SirA is a recently discovered regulator of DnaA in Bacillus subtilis whose synthesis is turned on at the start of sporulation. Here, we demonstrate that SirA contacts DnaA at a patch of 3 residues located on the surface of domain I of the replication initiator protein, corresponding to the binding site used by two unrelated regulators of DnaA found in other bacteria. We show that the interaction of SirA with domain I inhibits the ability of DnaA to bind to the origin of replication. DnaA mutants containing amino acid substitutions of the 3 residues are functional in replication initiation but are immune to inhibition by SirA.