Abstract <p>This study aimed to investigate the regulatory mechanisms by which endophytic bacteria influence secondary metabolite biosynthesis in broccoli (<i>Brassica oleracea</i> L. var. <i>italica</i> Planch) hairy roots, and successfully isolated and identified an endophytic strain, <i>Microbacterium liquefaciens</i> J1. The bacterium was inoculated on day 18 of hairy root culture (late proliferation stage), and the experiments were conducted based on the previously established co-culture system. By integrating transcriptome sequencing, qPCR validation, and dynamic physiological analyses, we found that a transient burst of superoxide anions (<InlineEquation ID="IEq1"> <EquationSource Format="TEX">\({\text{O}}_{2}^{{\bullet - }}\)</EquationSource> <!--PlntPhys2660082Lan-m1--> </InlineEquation>) occurred in the hairy roots 6 h after inoculation, accompanied by a rapid increase in antioxidant enzyme activities such as superoxide dismutase (SOD) and ascorbate peroxidase (APX), while hydrogen peroxide (H₂O₂) levels were precisely regulated to a new steady state. By 24 h, both glutathione (GSH) content and the GSH/GSSG ratio increased significantly, and the accumulation of glucoraphanin (GRA) and sulforaphane (SF) rose markedly. Further virus-induced gene silencing (VIGS) experiments showed that silencing either the glutathione synthase gene (<i>BoGSH1</i>) or the reductase gene (<i>BoGR</i>) led to a collapse of redox homeostasis, converting beneficial microbial signaling into sustained oxidative stress and completely suppressing GRA and SF biosynthesis. This study demonstrates that the endophyte <i>M. liquefaciens</i> activates the GSH metabolic pathway by triggering a controlled oxidative stress, driving cellular redox homeostasis toward a more reduced state and thereby promoting glucosinolate biosynthesis. We found that an intact GSH metabolic pathway may be a necessary prerequisite for symbiotic signal transduction, providing a new target for leveraging beneficial microbes to regulate crop quality.</p>