The initiation of bacterial chromosome DNA replication and its regulation are critical events. DnaA is essential for initiation of DNA replication and is conserved throughout bacteria. In Escherichia coli, hydrolysis of ATP-DnaA is promoted by Hda through formation of a ternary complex with DnaA and DnaN, ensuring the timely inactivation of DnaA during the replication cycle. In Bacillus subtilis, YabA also forms a ternary complex with DnaA and DnaN, and negatively regulates the initiation step of DNA replication. However, YabA shares no structural homology with Hda and the regulatory mechanism itself has not been clarified. Here, in contrast to Hda, we observed that dnaA transcription was stable during under- and overexpression of YabA. ChAP-chip assays showed that the depletion of YabA did not affect DNA binding by DnaA. On the other hand, yeast two-hybrid analysis indicated that the DnaA ATP-binding domain interacts with YabA. Moreover, mutations in YabA interaction-deficient mutants, isolated by yeast two-hybrid analysis, are located at the back of the ATP-binding domain, whereas Hda is thought to interact with the ATP-binding pocket itself. The introduction into B. subtilis of a dnaA(Y144C) mutation, which disabled the interaction with YabA but did not affect interactions either with DnaA itself or with DnaD, resulted in over-initiation and asynchronous initiation of replication and disabled the formation of YabA foci, further demonstrating that the amino acid on the opposite side to the ATP-binding pocket is important for YabA binding. These results indicate that YabA indeed regulates the initiation of DNA replication by a different mechanism from that used by Hda in the E. coli RIDA system. Interestingly, all DnaA mutants deficient in YabA binding also displayed reduced DnaD binding in yeast two-hybrid assays, suggesting that YabA can inhibit replication initiation through competitive inhibition of DnaD binding to DnaA.