background: Bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) poses a severe threat to kiwifruit production. Because of the insufficient efficacy and environmental safety of available treatments, novel antibacterial agents should be urgently developed. Antimicrobial peptides (AMPs) can be used as antimicrobials for disease control. In this study, we designed a novel AMP, Jelleine-Ic, and evaluated its antibacterial activity and mechanism of action against Psa. results: Jelleine-Ic with a half-maximal effective concentration of 1.67 μg/mL exhibited stronger antibacterial activity than did parent Jelleine-I. Jelleine-Ic targeted the Psa membrane, increased membrane permeabilization and dissipated membrane potential, resulting in calcium leakage. Electron microscopy revealed that Jelleine-Ic disrupted cell morphology and caused intracellular alterations. Moreover, this AMP penetrated the cell membrane, bound to DNA, and reduced the expression of genes related to DNA replication and repair. Jelleine-Ic also reduced esterase activity and induced intracellular reactive oxygen species generation. This peptide inhibited the development of Psa canker. The control efficiency of Jelleine-Ic against Psa in the leaf discs and leaves of kiwifruit was 81.83% and 70.53%, respectively, which was superior to that of the commercial agricultural streptomycin. Furthermore, Jelleine-Ic upregulated the expression of kiwifruit defense genes (PR-10 and WRKY70a). conclusions: Jelleine-Ic effectively controls Psa in vitro and in vivo, and may be developed as a bactericide for plant disease control. © 2023 Society of Chemical Industry.