Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139 has antagonist activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. Its biosynthesis gene cluster (ste) has been identified. In this study, gene ste17 was expressed in Escherichia coli BL21 and the recombinant protein was purified. With CTP and alpha-D-glucose-1-phosphate as substrates, the recombinant Ste17 protein was found capable of catalyzing the production of CDP-D-glucose and pyrophosphate, demonstrating its identity as an alpha-D-glucose-1-phosphate-cytidylyltransferase (CDP-D-glucose synthase). To investigate the function of ste17 in Ebosin biosynthesis, the gene was disrupted with a double crossover via homologous recombination. The monosaccharide composition of exopolysaccharide (EPS) produced by the mutant Streptomyces sp. 139 (ste17 (-)) was found significantly altered from that of Ebosin, with glucose becoming undetectable. This gene knockout also negatively affected the antagonist activity for IL-1R of EPS. These results indicate that the CDP-D: -glucose synthase encoded by ste17 gene is involved in the formation of nucleotide sugar (CDP-D-glucose) as glucose precursor in Ebosin biosynthesis.