Coronatine (COR) can enhance resistance to P. syringae pv. mori in the mulberry to avoid the outbreak of Mulberry Bacterial Blight. In order to analyze the defensive mechanism, metabolic responses of mulberry leaves to COR were examined by gas chromatography-mass spectrometry and biochemical analysis. Antimicrobial activities of metabolites against P. syringae in vitro were also detected by colorimetric assay. Metabolic profiles revealed that 0.01 μM COR induced changes of various metabolites including organic acids, sugars, alcohols, ketones, phenols, hydrocarbons, and nitrogen or sulfur compounds. In particular, sugars, organic acids, phenols, flavonoids, and nitrogen or sulfur compounds increased significantly in the COR-treated leaves. Biochemical analysis also confirmed above... findings. 1H-indole-3-acetic acid and abscisic acid were increased to regulate the signaling pathways; meanwhile, hydrogen peroxide and nitric oxide were induced to participate in the metabolic changes as signaling molecules. Concurrently, the metabolites showed antimicrobial activities against P. syringae. These results indicated that COR could increase contents of antimicrobial compounds by modulating the metabolic process to resist the pathogens entering into the plant tissues and prevent the outbreak of Mulberry Bacterial Blight.