Seed development is programmed by expression of many genes in plants. Seed maturation is an important developmental process to soybean seed quality and yield. DNA methylation is a major epigenetic modification regulating gene expression. However, little is known about the dynamic nature of DNA methylation and its effects on gene expression during plant development. Through whole-genome bisulfite sequencing, we showed that DNA methylation went through dynamic changes during seed maturation. An average of 66% CG, 45% CHG and 9% CHH contexts was methylated in cotyledons. CHH methylation levels in cotyledons changed greatly from 6% at the early stage to 11% at the late stage. Transcribed genes were approximately two-fold more likely to be differentially methylated than non-transcribed genes. We identified 40, 66 and 2136 genes containing differentially methylated regions (DMRs) with negative correlation between their expression and methylation in the CG, CHG and CHH contexts, respectively. The majority of the DMR genes in the CHH context were transcriptionally down-regulated as seeds mature: 99% of them during early maturation were down-regulated, and preferentially associated with DNA replication and cell division. The results provide novel insights into the dynamic nature of DNA methylation and its relationship with gene regulation in seed development.