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Glycine max L. accumulates a large amount of isoflavonoid compounds, which is beneficial for plant defense, plant-microbe symbiotic interactions, and human health. Several CYP450 subfamily genes are involved in the flavonoid biosynthetic pathway in plants. In the present study, we found 24 CYP82 subfamily genes were differentially expressed in various tissues of soybean, in Phytophthora sojae-infected soybean varieties and in soybean hairy roots treated with cell wall glucan elicitor. Six of them (GmCYP82A2, GmCYP82A3, GmCYP82A4, GmCYP82A23, GmCYP82C20 and GmCYP82D26) were co-expressed with other known isoflavonoid pathway genes in soybean. Their enzymatic activity in yeast feeding assays showed that only GmCYP82D26 was able to convert naringenin to daidzein with both aryl migration and dehydration function. When GmCYP82D26 was over-expressed in soybean hairy roots, the contents of the two major isoflavonoid aglycones in soybean (daidzein and genistein) were reduced, but total flavonoids were not affected. When GmCYP82D26 was suppressed by RNAi in the hairy roots, daidzein content was decreased but genistein content was increased, with unchanged total flavonoid content. GmCYP82D26 was found to be localized in the endoplasmic reticulum at subcellular level when transiently expressed in tobacco leaf epidermis. GmCYP82D26 gene was preferentially expressed in roots, with low expression level in other tissues in soybean. Homology modeling and molecular docking showed that GmCYP82D26 could form hydrogen bond with both HEM and naringenin at C5–OH and C4 carbonyl. All these results indicated that GmCYP82D26 possesses new and dual enzymatic activity, which bridges the two branches (daidzein and genistein branch) of isoflavonoid pathway in soybean.