Quercus variabilis (Q. variabilis), a significant broadleaf species used in afforestation across high, sandy, and mountainous regions, presents unique challenges for transplantation. This species is characterized by its slow above-ground growth and rapid taproot development, which suppresses the proliferation of lateral and fibrous roots, negatively impacting post-transplant survival. Research indicates that targeted root pruning—specifically, the removal of one-third of the roots—promotes the development of lateral roots in these seedlings. This study involved pruning the root systems of Q. variabilis and assessing the subsequent root development in comparison to an unpruned control group. Our analysis, which included transcriptome sequencing and plant hormone metabolism assays conducted at 2, 12, and 25 days post-pruning, yielded 126.02 Gb of clean data and identified 7662 differentially expressed genes (DEGs). These genes were primarily enriched in the plant hormone signal transduction pathway. Further investigation of this pathway, along with hormone content measurements, elucidated the mechanisms that contribute to enhanced root growth following pruning. Additionally, through a weighted correlation network analysis (WGCNA), we identified 20 key genes that are instrumental in promoting root development in Q. variabilis saplings. This research advances the theoretical framework for forestry seedling production and afforestation, laying the groundwork for scientifically informed vegetation restoration techniques.