DNMT1 is a DNA methyltransferase that catalyzes and maintains methylation in CpG dinucleotides. It blocks the entrance of DNA into the catalytic pocket via the replication foci targeting sequence（RFTS） domain. Recent studies have shown that an H3-tail-conjugated two-mono-ubiquitin mark（H3Ub2） activates DNMT1 by binding to the RFTS domain. However, the activation mechanism of DNMT1 remains unclear. In this work, we combine various sampling methods of extensive simulations, including conventional molecular dynamics, Gaussian-accelerated molecular dynamics, and coarse-grained molecular dynamics, to elucidate the activation mechanism of DNMT1. Geometric and energy analyses show that binding of H3Ub2 to the RFTS domain of DNMT1 results in the bending of the α4-helix in the RFTS domain at approximately 30°–35°, and the RFTS domain rotates ～20°anti-clockwise and moves ～3 ? away from the target recognition domain（TRD）. The hydrogen-bonding network at the RFTSTRD interface is significantly disrupted, implying that the RFTS domain is dissociated from the catalytic core, which contributes to activating the auto-inhibited conformation of DNMT1. These results provide structural and dynamic evidence for the role of H3Ub2 in regulating the catalytic activity of DNMT1.