Update publication information. Listeria monocytogenes is a foodborne pathogen, with relatively low incidence but high case-fatality. Phytochemicals have been recognized as a promising antimicrobial agent as an alternative to synthetic chemicals due to their safety and high efficacy with multi-target sites. This study identified and characterized a novel antibacterial agent, dehydrocorydaline, in Corydalis turschaninovii rhizome using HPLC-LTQ-Orbitrap-HRMS and its antibacterial effect with lowest MIC (1 mg/mL) and MBC (2 mg/mL) values. In addition, in vitro growth kinetic assay, cytoplasmic nucleic acid and protein leakage assay and observation of morphological changes in bacterial cells supported the strong antibacterial activity. Herein, quantitative proteomic analysis was performed to investigate the molecular mechanism of antibacterial action of dehydrocorydaline against L. monocytogenes. Taking together with proteomic results, we found that dehydrocorydaline influenced on the global proteomic alteration of L. monocytogenesis associated with carbohydrate metabolism, energy metabolism, cell wall synthesis, and bacterial motility, suggesting multiple target sites of dehydrocorydaline to inactivate bacterial growth. This study demonstrated that dehydrocorydaline has potential as a natural and effective antibacterial agent with multi-target sites in pathogenic bacteria, and provides the basis for development of a new class of antibacterial agent.