<span class="paragraphSection"><div class="boxTitle">Abstract</div>DNMT1 is a methyltransferase that restores 5-methylcytidine marks on newly replicated DNA and is required for maintaining epigenetic inheritance. Using Halo-tagged DNMT1 and highly inclined thin illumination (HiLo) microscopy, we show that DNMT1 mobility in living human cells changes under a variety of conditions. DNMT1 molecules become increasingly bound to chromatin in the S phase of the cell cycle, but surprisingly only ∼ 12% chromatin-bound DNMT1 is sufficient to maintain DNA methylation. Upon treatment with small molecule inhibitors, GSK-3484862 (GSK), 5-azacytidine (5-azaC) and decitabine (5-aza-deoxyC), <span style="font-style: italic;">in vivo</span> DNMT1 dynamics are greatly altered. Unexpectedly, treatment of cells with GSK, a non-covalent inhibitor, causes binding of DNMT1 to chromatin similar to that observed upon treatment with 5-azaC and decitabine, covalent inhibitors. 5-azaC inhibition of DNMT1 dynamics occurs during the S phase of the cell cycle. Unexpectedly, mutations in the disordered, Asp- and Glu-rich <span style="font-style: italic;">N</span>-terminal region of DNMT1 dramatically decrease its mobility and increase chromatin binding. Collectively, our work using live cell single molecule imaging quantifies the molecular dynamics of DNMT1 and how this relates to its function under physiological conditions and upon drug treatment. Understanding the dynamics of DNMT1 <span style="font-style: italic;">in vivo</span> provides a framework for developing better therapeutics that target DNMT1.</span>