We studied how the interaction between HindIII endonuclease and dsDNA is affected by the single-base modification of the latter by a single-molecule kinetic assay. For a comparative study of chemical modifications, we measured the binding and unbinding rates of the HindIII-DNA complex for normal dsDNA, methylated DNA, and hydroxymethylated DNA. We found that methylation of DNA at the recognition site results in a large increase in the unbinding rate due to the steric effect, which is consistent with the standard free energy change in the transition state. On the contrary, methylation minimally affects the binding rate, as simultaneous increases in the activation energy and the pre-exponential factor compensate for each other.