Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G·T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G·C pair. TDG cleaves the N-glycosylic bond of dT and some other nucleotides, including 5-substituted 2′-deoxyuridine analogs, once they have been flipped from the helix into its active site. We examined the role of two strictly conserved residues; Asn140, implicated in the chemical step, and Arg275, implicated in nucleotide flipping. The N140A variant binds substrate DNA with the same tight affinity as wild-type TDG, but it has no detectable base excision activity for a G·T substrate, and its excision rate is vastly diminished (by ∼104.4-fold) for G·U, G·FU, and G·BrU substrates. Thus, Asn140 does not contribute substantially to substrate binding but is essential for the chemical step, where it stabilizes the transition state by ∼6 kcal/mol (compared with 11.6 kcal/mol stabilization provided by TDG overall). Our recent crystal structure revealed that Arg275 penetrates the DNA minor groove, filling the void created by nucleotide flipping. We found that the R275A and R275L substitutions weaken substrate binding and substantially decrease the base excision rate for G·T and G·BrU substrates. Our results indicate that Arg275 promotes and/or stabilizes nucleotide flipping, a role that is most important for target nucleotides that are relatively large (dT and bromodeoxyuridine) and/or have a stable N-glycosylic bond (dT). Arg275 does not contribute substantially to the binding of TDG to abasic DNA product, and it cannot account for the slow product release exhibited by TDG.