l-Fucose, 6-deoxy-l-galactose, is a key component of many important glycoconjugates including the blood group antigens and the LewisX ligands. The biosynthesis of GDP-l-fucose begins with the action of a dehydratase that converts GDP-d-mannose into GDP-4-keto-6-deoxy-mannose. The enzyme GDP-fucose synthase, GFS, (also known as GDP-4-keto-6-deoxy-d-mannose epimerase/reductase, GMER) then converts GDP-4-keto-6-deoxy-d-mannose into GDP-l-fucose. The GFS reaction involves epimerizations at both C-3′′ and C-5′′ followed by an NADPH-dependent reduction of the carbonyl at C-4. This manuscript describes studies that elucidate the order of the epimerization steps and the roles of the active site acid/base residues responsible for the epimerizations. An active site mutant, Cys109Ser, produces GDP-6-deoxy-d-altrose as its major product indicating that C-3′′ epimerization occurs first and premature reduction of the GDP-4-keto-6-deoxy-d-altrose intermediate becomes competitive with GDP-l-fucose production. The same mutation results in the appearance of a kinetic isotope effect when [3′′-2H]-GDP-6-deoxy-4-keto-mannose is used as a substrate. This indicates that Cys109 is the base responsible for the deprotonation of the substrate at C-3′′. The Cys109Ser mutant also catalyzes a rapid wash-in of solvent derived deuterium into the C-5′′ position of GDP-fucose in the presence of NADP+. This confirms the order of epimerizations and the role of Cys109. Finally, the inactive His179Gln mutant readily catalyzes the wash-out of deuterium from the C-3′′ position of [3′′-2H]-GDP-6-deoxy-4-keto-mannose. Together these results strongly implicate an ordered sequence of epimerizations (C-3′′ followed by C-5′′) and suggest that Cys109 acts as a base and His179 acts as an acid in both epimerization steps.