Background: Poplar is frequently attacked by herbivorous insects, including the Asian gypsy moth, Lymantria dispar. Here, we combined metabolomic and transcriptomic analysis to identify key genes and metabolites involved into the molecular mechanism of defensive enhancement against L. dispar herbivory on poplar. Results: The 3666 differentially expressed genes (DEGs, 1,799 up-regulated and 1,867 down-regulated) and 1,171 DEGs (695 up-regulated and 476 down-regulated) were specific in L. dispar herbivory and mechanical wounding, respectively. Moreover, the 9,108 and 7,656 ions were detected while 636 and 531 different ions were obtained using positive (pos) mode and negative (neg) mode, respectively. Among these ions, the 33 and 7 different ions were specific in L. dispar herbivory and mechanical wounding in pos mode while 46 and 4 different ions in L. dispar herbivory and mechanical wounding in neg mode, respectively. The 3,666 specific DEGs in L. dispar herbivory group were classified into phenylpropanoid and flavonoid secondary metabolism pathways by comprehensive networks between transcriptomes and metabolomes. Conclusions: The current findings greatly improve our understanding of the induced defensive response in poplar plants against L. dispar infestation, and will contribute to develop insect-resistance poplar varieties.