The first enzyme in the formation of GDP-L-fucose from GDP-D-mannose, which forms a GDP-4-keto sugar intermediate, was purified to homogeneity from cell extracts of Klebsiella pneumoniae. During purification, the enzyme was found to be highly activated by NADP. It was proven that the pyridine nucleotide coenzyme of the enzyme was NADP, not NAD, which differs from previously accepted information. NAD had no effect on enzyme activity. The product of the enzyme reaction with NADP as coenzyme was separated from other nucleotides by high-performance liquid chromatography, and using ion spray liquid chromatography/mass spectrometry the mass was determined for the first time, as 587, which is same as the calculated mass of GDP-4-keto-6-deoxy-D-mannose.