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<i>Taxus</i>, a vital source of the anticancer drug paclitaxel, grapples with a pronounced supply–demand gap. Current efforts to alleviate the paclitaxel shortage involve expanding <i>Taxus</i> cultivation through cutting propagation. However, traditional cutting propagation of <i>Taxus</i> is difficult to root and time-consuming. Obtaining the roots with high paclitaxel content will cause tree death and resource destruction, which is not conducive to the development of the <i>Taxus</i> industry. To address this, establishing rapid and efficient stem rooting systems emerges as a key solution for <i>Taxus</i> propagation, facilitating direct and continuous root utilization. In this study, <i>Agrobacterium rhizogenes</i> were induced in the 1–3-year-old branches of <i>Taxus</i> × <i>media</i> Rehder, which has the highest paclitaxel content. The research delves into the rooting efficiency induced by different <i>A. rhizogenes</i> strains, with MSU440 and C58 exhibiting superior effects. Transcriptome and metabolome analyses revealed <i>A. rhizogenes</i>’ impact on hormone signal transduction, amino acid metabolism, zeatin synthesis, and secondary metabolite synthesis pathways in roots. LC-MS-targeted quantitative detection showed no significant difference in paclitaxel and baccatin III content between naturally formed and induced roots. These findings underpin the theoretical framework for <i>T. media</i> rapid propagation, contributing to the sustainable advancement of the <i>Taxus</i> industry.