Nodulation is crucial for biological nitrogen fixation (BNF) in legumes, but the molecular mechanisms underlying BNF have remained elusive. Here, we cloned a candidate gene underlying a major nodulation quantitative trait locus in soybean (Glycine max), INCREASING NODULE SIZE1 (GmINS1). GmINS1 encodes a cell wall β-expansin and is expressed primarily in vascular bundles, along with cortical and parenchyma cells of nodules. Four single-nucleotide polymorphisms distinguishing the two parents were found in the GmINS1 promoter region. Among them, single-nucleotide polymorphism A/C has a significant effect on GmINS1 expression in the parental genotype P2, based on β-glucuronidase activity and promoter deletion analysis. The expression of GmINS1 and the P2 genotype promoter was strongly associated with nodule development, not only in the parents but also in 40 progeny lines and 40 genotypes selected from a soybean core collection. Overexpression of GmINS1 resulted in increases in the number, biomass, infection cell abundance, and nitrogenase activity of large nodules and subsequently changed the nitrogen content and biomass of soybean plants. GmINS1 suppression via RNA interference had the opposite effect. Double suppression of GmEXPB2 and GmINS1 dramatically inhibited soybean nodulation. Our results reveal that GmINS1 is a critical gene in nodule development and that GmEXPB2 and GmINS1 synergistically control nodulation in soybean. Our findings shed light on the genetic basis of soybean nodulation and provide a candidate gene for optimizing BNF capacity through molecular breeding in soybean.