The trisaccharide unit in lipopolysaccharide of Bordetella pertussis is important for immune recognition, but how the rare sugar 2-acetamido-4-methylamino-fucose in the trisaccharide unit is synthesized and transferred to the core of lipopolysaccharide in B. pertussis remains unclear. In this study, we demonstrated that the genes bplF, bplG, bplL and bplI are involved in the synthesis and transfer of 2-acetamido-4-methylamino-fucose to the core of lipopolysaccharide in B. pertussis. These genes were overexpressed with various combination in a recombinant E. coli strain which can synthesize B. pertussis core-lipid A. Lipopolysaccharides were isolated from these strains and analyzed by using SDS-PAGE, ion chromatography and NMR. Based on these analyses, the mechanism of biosynthesis and transfer of 2-acetamido-4-methylamino-l-fucose to the core-lipid A in B. pertussis is proposed.