Abstract Populus canker is a widespread disease that seriously threatens the survival of trees. Phytohormones are considered as effective chemical molecules improving plant resistance to various diseases. Ethylene is an important phytohormone that is extensively involved in the regulation of plant growth, development and stress responses, but how ethylene and ethylene signaling regulates defense responses in woody plants is still unclear. Here, we showed that ethylene positively regulates the responses of poplar to canker caused by the hemibiotrophic fungus Dothiorella gregaria. Treatment of Populus tomentosa with 1-aminocyclopropane-1-carboxylic acid (ACC, the biosynthetic precursor of ethylene) significantly enhanced disease resistance, accompanied by the induction of pathogen-related protein (PR) gene expression and H2O2 accumulation. Blocking ethylene biosynthesis using Aminoethoxyvinyl glycine (AVG, a specific inhibitor of ethylene biosynthesis) repressed the disease resistance. Overexpression of the ethylene biosynthesis gene PtoACO7 in Populus tomentosa promoted defense responses and disease resistance. Furthermore, we demonstrated that the ethylene-induced defense response is independent of the salicylic acid pathway, but needs ROS signaling. ACC or PtoACO7 overexpression induced expressions of PtoRbohD/RbohF, which encode NADPH oxidases, and elevated H2O2 levels in poplar. Inhibition of the NADPH oxidase compromised ethylene-induced disease resistance and PR gene expressions, while H2O2 application could completely rescue the AVG-caused disease hypersensitivity. Therefore, the involvement of ethylene in disease resistance is done by activation of PR gene expressions and ROS production. Our results also showed that modifying ethylene biosynthesis or its signaling pathway has a great potential for improving disease resistance in woody plants. This article is protected by copyright. All rights reserved.