The dTDP-glucose 4,6-dehydratase catalyzed conversion of dTDP-glucose to dTDP-4-keto-6-deoxyglucose occurs in three sequential chemical steps: dehydrogenation, dehydration, and rereduction. The enzyme contains the tightly bound coenzyme NAD(+), which mediates the dehydrogenation and rereduction steps of the reaction mechanism. In this study, we have determined that Asp135 and Glu136 are the acid and base catalysts, respectively, of the dehydration step. Identification of the acid catalyst was performed using an alternative substrate, dTDP-6-fluoro-6-deoxyglucose (dTDP-6FGlc), which undergoes fluoride ion elimination instead of dehydration, and thus does not require protonation of the leaving group. The steady-state rate of conversion of dTDP-6FGlc to dTDP-4-keto-6-deoxyglucose by each Asp135 variant was identical to that of wt, in contrast to turnover using dTDP-glucose where differences in rates of up to 2 orders of magnitude were observed. These results demonstrate Asp135's role in protonating the glucosyl-C6(OH) during dehydration. The base catalyst was identified using a previously uncharacterized, enzyme-catalyzed glucosyl-C5 hydrogen-solvent exchange reaction of product, dTDP-4-keto-6-deoxyglucose. Base catalysis of this exchange reaction is analogous to that occurring at C5 during the dehydration step of net catalysis. Thus, the decrease in the rate of catalysis ( approximately 2 orders of magnitude) of the exchange reaction observed with Glu136 variants demonstrates this residue's importance in base catalysis of dehydration.