DNA methylation plays an essential role in transcriptional control of organismal development in epigenetics, from turning off a specific gene to inactivation of entire chromosomes. While the biological function of DNA methylation is becoming increasingly clear, the mechanism of methylation-induced gene regulation is still poorly understood. Through single-molecule force experiments and simulation we investigated the effects of methylation on strand separation of DNA, a crucial step in gene expression. Molecular force assay and single-molecule force spectroscopy revealed a strong methylation dependence of strand separation. Methylation is observed to either inhibit or facilitate strand separation, depending on methylation level and sequence context. Molecular dynamics simulations provided a detailed view of methylation effects on strand separation, suggesting the underlying physical mechanism. According to our study, methylation in epigenetics may regulate gene expression not only through mechanisms already known but also through changing mechanical properties of DNA.