Galactofuranose (Galf) is the five membered ring form of galactose. It is found on the cell wall and surface of many pathogens including Mycobacterium tuberculosis, Aspergillus fumigatus, Leishmania major, and Trypanosoma cruzi. Galf has been implicated in pathogenesis in these organisms; thus the biosynthetic pathway of Galf is a target for drug design. Galf is synthesized by the enzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). Solving the mechanism and structure of UGMs will aid in the development of specific inhibitors against these enzymes. Herein we present the detailed functional analysis of UGMs from A. fumigatus, T. cruzi, and L. major. The mechamism and structure these eukaryotic UGMs were examined by steady-state kinetics, rapid-reaction kinetics, trapping of reaction intermediates, fluorescence anisotropy, and X-ray crystallography. The mechanism first involves reduction of the required flavin by NADPH, followed by UDP-Galp binding and subsequent SN2 attack by the flavin on galactose displacing UDP to form a flavin N5-C1 galactose adduct. Next, the galactose ring opens forming an iminium ion allowing isomerization to occur. Lastly, the product is released and UGM is available to bind another substrate or be reoxidized by molecular oxygen. The three-dimensional structure of A. fumigatus UGM was solved using X-ray crystallography in four conformations: oxidized in complex with sulfate ions, reduced, reduced in complex with UDP, and reduced in complex with UDP-Galp, giving valuable information on the unique features of eukaryotic UGMs including features important for oligomerization and for substrate binding. The novel mechanism and structure provide valuable information for the development of specific inhibitors of eukaryotic UGMs.