UDP-galactose mutase is a flavoenzyme that catalyzes the isomerization of UDP-galactopyranose into UDP-galactofuranose, a key step in the biosynthesis of important bacterial oligosaccharides. Several mechanisms for this unique ring-contraction have been proposed, one of them involving a putative 1,4-anhydrogalactopyranose as an intermediate in the reaction. The purpose of this study was to probe the mutase binding site with conformationally restricted analogues of its substrate. Thus, we describe the straightforward synthesis of two C-glycosidic UDP-galactose derivatives: analogue 1, presenting a galactose moiety locked in a bicyclic (1,4)B boat conformation, and UDP-C-Galf 2, where the galactose residue is locked in the conformation of the mutase substrate. The two molecules were found to be inhibitors of UDP-galactose mutase at levels depending on the redox state of the enzyme. Strong inhibition of the native enzyme, but a low one of the reduced mutase, were observed with UDP-C-Galf 2, whereas 1 displayed intermediate inhibition levels under both native and reducing conditions. These data provide evidence of a significant conformational difference of the mutase binding pocket in the reduced enzyme and in the native one, the enzyme switching from a low Galf-affinity state (reduced enzyme) to a very strong one (native enzyme). It is remarkable that the mutase binds the boat-locked analogue 1 with similar affinities in both its conformational states. These results support a mechanism involving the formation of 1,4-anhydrogalactopyranose as a low-energy intermediate. An alternative explanation would be that the distortion of the galactose moiety during the cycle contraction transiently brings the carbohydrate into a conformation close to a (1,4)B boat.