Abstract: Soybean（Glycine max） is the important economic crop, which can provide nutrients such as oil and protein for human beings. Nodule development is one of the most critical biological processes of soybean, which can help soybean fix nitrogen in the air and obtain nitrogen needed for growth and development. However, the epigenetic regulation mechanism of soybean nodule formation is still unclear. In this study, DNA methylome analysis was performed between soybean nodule and root by genome-wide bisulfite sequencing at a single base resolution. Nodules showed lower CG and CHG genome-wide DNA methylation than that in roots, but higher CHH methylation. Moreover, thousands of differential methylation regions（DMRs） were found, especially in TE and intergenic regions. In addition, gene body and upstream/downstream regions also contained a large number of DMRs, suggesting that these DMRs may be involved in the expression regulation of soybean nodule. Combined with transcriptome data, many DMRs were associated with differential expression genes, and these DMR-associated differential expression genes contained some important nodule-related genes, such as NIN, NF-YA, and SymRK. Taken together, our findings can explain the DNA methylation regulation of transcription difference between nodule and root of soybean.