Since the root nodules formation is regulated by specific and complex interactions of legume and rhizobial genes, there are still too many questions to be answered about the role of the genes involved in the regulation of the nodulation signaling pathway. The genetic and biological roles of the isoflavone-7-O-beta-glucoside 6″-O-malonyltransferase gene GsIMaT2 from wild soybean (Glycine soja) in the regulation of nodule and root growth in soybean (Glycine max) were examined in this work. The effect of overexpressing GsIMaT2 from G. soja on the soybean nodulation signaling system and strigolactone production was investigated. We discovered that the GsIMaT2 increased nodule numbers, fresh nodule weight, root weight, and root length by boosting strigolactone formation. Furthermore, we examined the isoflavone concentration of transgenic G. max hairy roots 10 and 20 days after rhizobial inoculation. Malonyldaidzin, malonylgenistin, daidzein, and glycitein levels were considerably higher in GsIMaT2-OE hairy roots after 10- and 20-days of Bradyrhizobium japonicum infection compared to the control. These findings suggest that isoflavones and their biosynthetic genes play unique functions in the nodulation signaling system in G. max. Finally, our results indicate the potential effects of the GsIMaT2 gene on soybean root growth and nodulation. This study provides novel insights for understanding the epistatic relationship between isoflavones, root development, and nodulation in soybean