The full-length cDNA of GDP-fucose transporter (GFT) was cloned from Chinese hamster ovary (CHO) 13D-35D cells. The predicted product consisted of 366 amino acids with 86% and 92% similarities to human GFT and mouse GFT, respectively. When synthetic small interfering RNA (siRNA) against CHO GFT was introduced into human antithrombin III (AT-III)-producing recombinant CHO cells, defucosylated AT-III increased by 30-40% after 3-d incubation. A GFT siRNA expression plasmid was also constructed to defucosylate recombinant AT-III. CHO cells, which were transfected with the siRNA expression plasmid, showed approximately 75% reduction of Golgi-GDP fucose, resulting in 10-40% increase in defucosylated AT-III.