The regulation of chromosomal replication is critical and the activation of DnaA by ATP binding is a key step in replication initiation. However, it remains unclear whether and how the process of ATP-binding to DnaA is regulated. Here, we show that DnaA can be acetylated, and its acetylation level varies with cell growth and correlates with DNA replication initiation frequencies in E. coli. Specifically, the conserved K178 in Walker A motif of DnaA can be acetylated and its acetylation level reaches the summit at the stationary phase, which prevents DnaA from binding to ATP or oriC and leads to inhibition of DNA replication initiation. The deacetylation process of DnaA is catalyzed by deacetylase CobB. The acetylation process of DnaA is mediated by acetyltransferase YfiQ, and nonenzymatically by acetyl-phosphate. These findings suggest that the reversible acetylation of DnaA ensures cells to respond promptly to environmental changes. Since Walker A motif is universally distributed across organisms, acetylation of Walker A motif may present a novel regulatory mechanism conserved from bacteria to eukaryotes.