Thymine DNA glycosylase (TDG) is a DNA repair enzyme that excises a variety of mismatched or damaged nucleotides (nts), e.g. dU, dT, 5fC and 5caC. TDG is shown to play essential roles in maintaining genome integrity and correctly programming epigenetic modifications through DNA demethylation. After locating the lesions, TDG employs a base-flipping strategy to recognize the damaged nucleobases, whereby the interrogated nt is extruded from the DNA helical stack and binds into the TDG active site. The dynamic mechanism of the base-flipping process at an atomistic resolution, however, remains elusive. Here, we employ the Markov State Model (MSM) constructed from extensive all-atom molecular dynamics (MD) simulations to reveal the complete base-flipping process for a G.T mispair at a tens of microsecond timescale. Our studies identify critical intermediates of the mispaired dT during its extrusion process and reveal the key TDG residues involved in the inter-state transitions. Notably, we find an active role of TDG in promoting the intrahelical nt eversion, sculpturing the DNA backbone, and penetrating into the DNA minor groove. Three additional TDG substrates, namely dU, 5fC, and 5caC, are further tested to evaluate the substituent effects of various chemical modifications of the pyrimidine ring on base-flipping dynamics.