L-Fucose (6-deoxy-L-galactose) is a major constituent of glycans and glycolipids in mammals. Fucosylation of glycans can confer unique functional properties and may be an economical way to manufacture L-fucose. Research can extract L-fucose directly from brown algae, or by enzymatic hydrolysis of L-fucose-rich microbial exopolysaccharides. However, these L-fucose production methods are not economical or scalable for various applications. We engineered an Escherichia coli strain to produce L-fucose. Specifically, we modified the strain genome to eliminate endogenous L-fucose and lactose metabolism, produce 2 '-fucosyllactose (2 '-FL), and to liberate L-fucose from 2 '-FL. This E. coli strain produced 16.7 g/L of L-fucose with productivity of 0.1 g center dot L-1 center dot h(-1) in a fed-batch fermentation. This study presents an efficient one-pot biosynthesis strategy to produce a monomeric form of L-fucose by microbial fermentation, making large-scale industrial production of L-fucose feasible.