Key message <p><i>PagC3H3</i>, encoding a <i>p</i>-coumarate 3-hydroxylase, catalyzes a critical step in lignin biosynthesis and confers enhanced insect resistance in poplar. Its overexpression increases lignin deposition, enhancing physical barrier formation against arthropod herbivory (e.g., reduced gypsy moth larval consumption). This provides a genetic engineering strategy for improving wood properties and biotic stress resilience.</p> Abstract <p>Lignin, a complex phenolic polymer crucial for structural integrity and biotic stress resistance in poplar, critically influences wood processing efficiency and ecological resilience. Targeted genetic manipulation of key enzymes like <i>p</i>-coumarate 3-hydroxylase (C3H) remains underexplored in commercial hybrids. In this study, we investigated the role of <i>PagC3H3</i> in lignin biosynthesis in the hybrid poplar <i>Populus alba</i> × <i>Populus glandulosa</i> (clone 84K). Through the overexpression and suppression of <i>PagC3H3</i> in poplar 84K, we observed that elevated <i>PagC3H3</i> expression significantly increases lignin content and enhances resistance to insect pests by enhancing physical barrier formation against arthropod herbivory, whereas RNAi-suppression lines exhibited reduced lignin and compromised growth. RNA-seq results showed that the expression of genes related to secondary cell wall components changed significantly, and 985 differentially expressed genes were co-regulated in the two transgenic lines, involving key transcription factors and structural genes for lignin synthesis and cell wall development. The results revealed the key role of <i>PagC3H3</i> in regulating lignin synthesis and cell wall development, and provided a new strategy for improving the wood properties and insect resistance of trees. This study not only improves the understanding of the molecular mechanism of lignin synthesis, but also provides a theoretical basis for the genetic improvement of poplar.</p>