Selenium (Se) is an essential micronutrient to humans. Se biofortification in crops provides an effective means to enhance dietary Se intake. However, the effects of Se treatment on plant growth and development remain to be fully studied. In this study, we applied Se to soybean plants grown hydroponically and in soil. We observed that selenate treatment increased nodule number and fresh weight. Furthermore, Se supplementation increased isoflavone daidzein accumulation in roots and nodules. An exogenous supply of daidzein but not kaempferol was found to increase nodule number and fresh weight significantly, suggesting a role of daidzein in the Se-induced nodulation in soybean plants. In addition, the total sugar levels in nodules, roots, and leaves were significantly enhanced following selenate treatment. Subsequently, the N-compounds were greatly increased after Se treatment in all tissues examined. Transcriptome analysis revealed that Se treatment altered various metabolic and cellular processes to affect nodulation and nodule development. Se treatment upregulated GmNIN2b, an important nodule organogenesis gene, as well as genes associated with sugar metabolism and ureide transport. Our study provides insights underlying the Se-induced nodulation in soybean plants and further documents that Se biofortification in soybean can simultaneously improve not only the essential nutrient content but also nodulation for biological nitrogen fixation, a critically important agronomic trait in soybean plants.