D-fucofuranose (D-Fucf) is a component of Escherichia coli O52 O antigen. This uncommon sugar is also the sugar moiety of the anticancer drug – gilvocarcin V produced by many streptomycetes. In E. coli O52, rmlA, rmlB, fcf1 and fcf2 were proposed in a previous study by our group to encode the enzymes of the dTDP-D-Fucf (the nucleotide-activated form of D-Fucf) biosynthetic pathway. In this study, Fcf1 and Fcf2 from E. coli O52 were expressed, purified and assayed for their respective activities. Novel product peaks from enzyme-substrate reactions were detected by capillary electrophoresis and the structures of the product compounds were elucidated by electro-spray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Fcf1 was confirmed to be a dTDP-6-deoxy-D-xylo-hex-4-ulopyranose reductase for the conversion of dTDP-6-deoxy-D-xylo-hex-4-ulopyranose to dTDP-D-fucopyranose (dTDP-D-Fucp), and Fcf2 a dTDP-D-Fucp mutase for the conversion of dTDP-D-Fucp to dTDP-D-Fucf. The Km of Fcf1 for dTDP-6-deoxy-D-xylo-hex-4-ulopyranose was determined to be 0.38 mM, and of Fcf2 for dTDP-D-Fucp to be 3.43 mM. The functional role of fcf1 and fcf2 in the biosynthesis of E. coli O52 O antigen were confirmed by mutation and complementation tests. This is the first time that the biosynthetic pathway of dTDP-D-Fucf has been fully characterized.