Legionaminic acid (Leg5,7Ac2) is a nonulosonic acid similar to sialic acid (Neu5Ac), which can be found in the extracellular glycoconjugates of several bacterial pathogens. Due to the similarity in stereochemistry of the two compounds, legionaminic acid has great potential in the production of pharmaceutical drugs. A novel biosynthetic pathway to produce legionaminic acid was created to overcome the limitations of organic synthesis. This is the first study involving the scale-up of legionaminic acid production by high cell density fermentation processes. In this work, fed-batch cultivations of recombinant Escherichia coli BRL04 were carried out in shake flasks and 5-L bioreactors. The final process was optimized by determining the effects of different carbon sources, induction temperatures, pH, dissolved oxygen (DO) content, induction optical density and N-acetylglucosamine (GlcNAc) feed rate on the production of legionaminic acid. Overall, results showed that the titer, yield and productivity for legionaminic acid production achieved relatively high levels, which were 5.53 g/L, 73.29% and 0.092 g/(Lh), respectively. It is hoped that this study accelerates research into the production of legionaminic acid for therapeutic treatments as well as for further study in glycobiology.