Over the past few decades, global maternal obesity prevalence has rapidly increased. This condition may induce long-lasting pathophysiological effects on either fetal or infant health that could be attributable to unknown unique changes in the umbilical blood composition. A total of 34 overweight/obese and 32 normal-weight pregnant women were recruited. Fifteen umbilical blood samples including 8 overweight/obese subjects and 7 normal weight women were sequenced using Targeted Bisulfite Sequencing technology to detect the average methylation level of cytosine and identify the differentially methylated region (DMR). GO and KEGG analyses were then employed to perform pathway enrichment analysis of DMR-related genes and promoters. Moreover, the mRNA levels of methylation-related genes histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) were characterized in the samples obtained from these two groups. Average methylated cytosine levels in both the CpG islands (CGI) and promoter significantly decreased in overweight/obese groups. A total of 1669 DMRs exhibited differences in their DNA methylation status between the overweight/obese and control groups. GO and KEGG analyses revealed that DMR-related genes and promoters were enriched in the metabolism, cancer and cardiomyopathy signaling pathways. Furthermore, the HDACs and DNMTs mRNA levels trended to decline in overweight/obese groups. Decreased methylated cytosine levels in overweight/obese women induce the gene expression activity at a higher level than in the control group. DMRs between these two groups in the fetal blood may contribute to the changes in gene transcription that underlie the increased risk of metabolic disorders, cancers and cardiomyopathy in their offspring.