DNA methylation to yield 5-methylcytosine (5mC) in CpG motifs plays a vital role in epigenetic regulation. However, deamination of 5mC results in canonical thymine (T) that requires methyl-CpG-binding domain protein 4 (MBD4) for repair. This important function has resulted in MBD4 being implicated in various human health disorders including MBD4-associated neoplasia syndrome and cancer resistance to 5-fluorouracil treatment. Nevertheless, the catalytic mechanism of MBD4 is poorly understood, with conflicting experimental observations resulting in multiple proposals. To provide atomic level structural details of the active site conformation and clarify the mechanistic pathway, this study uses a combination of adaptively biased molecular dynamics (abMD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations to map the MBD4 catalytic mechanism. Although our data indicate that the catalytic D560 residue is flexible in the active site, only one conformation facilitates 5mC excision. Despite some literature proposing the formation of a DNA−protein crosslinked intermediate, our modeling suggests catalysis is only viable through a deglycosylation mechanism that involves a water nucleophile attacking C1′ of T, with D560 activating the nucleophile and stabilizing the transition state and nucleobase departure facilitated by a network of hydrogen bonds. This proposal is fully consistent with experimental crystallographic, mutagenic, stereoscopic, and kinetic data, and aligns the MBD4 catalytic pathway with that characterized for several other monofunctional DNA glycosylases. By furthering our knowledge of MBD4 catalysis, this work will aid in the future development of treatments for MBD4-related genetic disorders and the rational design of transition state mimic inhibitors to enhance existing cancer therapies.