ABSTRACTThe Agr quorum sensing (QS) system is known to contribute to biofilm formation inListeria monocytogenes. Cinnamaldehyde, a natural food preservative, is considered an inhibitor of Agr-mediated QS inL. monocytogenes. However, the exact mechanism by which cinnamaldehyde acts on Agr remains unclear. In this study, we assessed the effects of cinnamaldehyde on the histidine kinase AgrC and the response regulator AgrA in the Agr system. AgrC kinase activity was not influenced by cinnamaldehyde, and binding between AgrC and cinnamaldehyde was not observed when microscale thermophoresis (MST) was performed, indicating that AgrC was not the target of cinnamaldehyde. AgrA is specifically bound to theagrpromoter (P2) to activate the transcription of the Agr system. However, AgrA-P2binding was prevented by cinnamaldehyde. The interaction between cinnamaldehyde and AgrA was further confirmed with MST. Two conserved amino acids, Asn-178 and Arg-179, located in the LytTR DNA-binding domain of AgrA, were identified as the key sites for cinnamaldehyde-AgrA binding by alanine mutagenesis and MST. Coincidentally, Asn-178 was also involved in the AgrA-P2interaction. Taken together, these results suggest that cinnamaldehyde acts as a competitive inhibitor of AgrA in AgrA-P2binding, which leads to suppressed transcription of the Agr system and reduced biofilm formation inL. monocytogenes.IMPORTANCEListeria monocytogenescan form biofilms on various food contact surfaces, posing a serious threat to food safety. Biofilm formation ofL. monocytogenesis positively regulated by the Agr quorum sensing system. Thus, an alternative strategy for controllingL. monocytogenesbiofilms is interfering with the Agr system. Cinnamaldehyde is considered an inhibitor of theL. monocytogenesAgr system; however, its exact mechanism of action is still unclear. Here, we found that AgrA (response regulator), rather than AgrC (histidine kinase), was the target of cinnamaldehyde. The conserved Asn-178 in the LytTR DNA-binding domain of AgrA was involved in cinnamaldehyde-AgrA and AgrA-P2binding. Therefore, the occupation of Asn-178 by cinnamaldehyde suppressed transcription of the Agr system and reduced biofilm formation inL. monocytogenes. Our findings could provide a better understanding of the mechanism by which cinnamaldehyde inhibitsL. monocytogenesbiofilm formation.