Tyvelose is a 3,6-dideoxyhexose found in the O-antigen of Yersinia pseudotuberculosis IVA and is the only member of this class of sugars to be produced directly from another 3,6-dideoxyhexose, paratose. The C-2 epimerization required for this conversion has been proposed to be catalyzed by CDP-d-tyvelose 2-epimerase. This enzyme is intriguing since it belongs to a group of epimerases, including the well-studied UDP-d-galactose 4-epimerase, that can invert unactivated stereocenters. To study the mechanism of this enzyme, we have cloned and expressed the tyv gene that encodes CDP-d-tyvelose 2-epimerase. The purified tetrameric protein contains approximately one equivalent of bound NAD+ per monomer and a small fraction of NADH. Four possible mechanisms involving NAD+ can be proposed for this enzyme; two involve oxidation at C-2 of the substrate, while the other two require oxidation at C-4. In a previous contribution, we presented preliminary data that supported a retro-aldol-type mechanism initiated by C-4 oxidation. However, this mechanism was refuted by further investigations, which revealed that the 4-fluoro analogue of CDP-d-paratose could be turned over by the enzyme. More importantly, the direct transfer of a deuterium from C-2 of the labeled substrate to the enzyme-bound NAD+ was observed by mass spectrometry. These results suggest that epimerization is in fact initiated by oxidation at C-2, followed by the transfer of the hydride from the transiently formed NADH to the opposite side of the 2-hexulose intermediate.