Isoflavonoids, predominantly found in legumes, are specialized metabolites with antioxidant properties that benefit both plant resilience and human health. Using metabolic genome-wide association studies (mGWAS), we identified the cytochrome P450 gene (Glyma.11g108300), GmIF6H1, as a key determinant of glycitein biosynthesis in soybean (Glycine max (L.) Merr.). Biochemical assays together with in planta stable-isotope tracing demonstrated that GmIF6H1 catalyzes the 6-hydroxylation of daidzein, establishing a previously unrecognized and predominant biosynthetic route for glycitein. A single amino-acid substitution in GmIF6H1 accounts for the domestication-associated reduction of glycitein-type isoflavonoids. Upon Phytophthora sojae infection, (malonyl)glycitins undergo sustained deglycosylation to release glycitein aglycone, underscoring its defensive role. Strikingly, both loss- and gain-of-function alleles increase susceptibility to P. sojae, indicating that precise tuning of GmIF6H1 expression is essential for effective resistance. Metabolite profiling further reveals complementary daidzein-centered defense strategies: glycitein-type isoflavonoids (via daidzein 6-hydroxylation) function as phytoanticipins, whereas glyceollins (via daidzein 2’-hydroxylation) act as inducible phytoalexins. Together,these findings clarify the biosynthetic origin of the glycitein and underscore the synergistic action of glycitein and glyceollins in pathogen resistance,offering new opportunities for engineering disease-resilient soybean cultivars.