Validamycin A (VMA) is an aminoglycoside antibiotic used to control rice sheath blight. Although it has been reported that VMA can induce the plant defense responses, the mechanism remains poorly understood. Here, we found that reactive oxygen species (ROS) bursts and callose deposition in Arabidopsis thaliana, rice (Oryza sativa L.), and wheat (Triticum aestivum L.) were induced by VMA and were most intense with 10 μg of VMA per milliliter at 24 h. Moreover, we showed that VMA induced resistance against Pseudomonas syringae, Botrytis cinerea, and Fusarium graminearum in Arabidopsis leaves, indicating that VMA induces broad-spectrum disease resistance in both dicots and monocots. In addition, VMA-mediated resistance against P. syringae was not induced in NahG transgenic plants, was partially decreased in npr1 mutants, and VMA-mediated resistance to B. cinerea was not induced in npr1, jar1, and ein2 mutants. These results strongly indicated that VMA triggers plant defense responses to both biotrophic and necrotrophic pathogens involved in salicylic acid (SA) and jasmonic acid/ethylene (JA/ET) signaling pathways and is dependent on NPR1. In addition, transcriptome analysis further revealed that VMA regulated the expression of genes involved in SA, JA/ET, abscisic acid (ABA), and auxin signal pathways. Taken together, VMA induces systemic resistance involving in SA and JA/ET signaling pathways and also exerts a positive influence on ABA and auxin signaling pathways. Our study highlights the creative application of VMA in triggering plant defense responses against plant pathogens, providing a valuable insight into applying VMA to enhance plant resistance and reduce the use of chemical pesticides.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.