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Poplars can be harmed by poplar canker. Inoculation with mycorrhizal fungi can improve the resistance of poplars to canker, but the molecular mechanism is still unclear. In this study, an aseptic inoculation system of <i>L. bicolor</i>–<i>P. trichocarpa</i>–<i>B. dothidea</i> was constructed, and transcriptome analysis was performed to investigate regulation by <i>L. bicolor</i> of the expression of genes in the roots of <i>P. trichocarpa</i> during the onset of <i>B. dothidea</i> infection, and a total of 3022 differentially expressed genes (DEGs) were identified. Weighted correlation network analysis (WGCNA) was performed on these DEGs, and 661 genes’ expressions were considered to be affected by inoculation with <i>L. bicolor</i> and <i>B. dothidea</i>. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these 661 DEGs were involved in multiple pathways such as signal transduction, reactive oxygen metabolism, and plant-pathogen interaction. Inoculation with <i>L. bicolor</i> changed the gene expression pattern of the roots, evidencing its involvement in the disease resistance response of <i>P. trichocarpa</i>. This research reveals the mechanism of <i>L. bicolor</i> in inducing resistance to canker of <i>P. trichocarpa</i> at the molecular level and provides a theoretical basis for the practical application of mycorrhizal fungi to improve plant disease resistance.