A recombinant Escherichia coli strain was developed to produce  guanosine 5'-diphosphate(GDP)-L-fucose, a donor of L-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides.  GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-L-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β-D-thioglucopyranoside(IPTG). A maximum GDP-L-fucose concentration of 38.9±0.6 mg/L was obtained in a glucose-limited fed-batch fermentation and it was enhanced further by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2±0.5 mg/L GDP-L-fucose concentration under the same fermentation condition.