To investigate the function of the GmSBT1 gene in nitrogen fixation in symbiosis between rhizobia and host plants, bioinformatics analysis, spatiotemporal expression localization, GUS staining localization and gene silencing were used to investigate its role in soybean rhizobia symbiosis. The results showed that the gene was specifically induced by rhizobia and only expressed at high levels in the rhizobia, possibly in the rhizobia cortex and bacterial cells. RNAi interference significantly reduced the aboveground fresh weight, nodule weight, and nitrogenase activity. This indicates that the GmSBT1 protein plays an important role in the formation and development of nodules, as well as nitrogen fixation in nodules.