Glycosylases specifically recognize and flip their target base out of the DNA helix into the enzyme's active site. Our simulations show that a partially flipped state, already present in free DNA carrying a T:G mispair, becomes the more probable state compared to the closed state after binding of thymine DNA glycosylase (TDG). Paired thymine (T:A) or methyl-cytosine (mC:G) does not exhibit a partially flipped state in free or complexed DNA. Important enzyme-DNA interactions exhibit significant strength in the intrahelical and extrahelical TDG-DNA complexes. The computed binding free energy differences suggest these interactions account for the stabilization of the partially flipped state, thereby driving the T:G mispair toward base flip. In the fully flipped state, the cognate base thymine is significantly better accommodated in the enzyme's active site than noncognate bases are, suggesting the hydrolysis step as the last of several stages at which base recognition can be achieved.