Heparosan as an acidic polysaccharide is mainly applied for heparin biosynthesis and drug delivery. Escherichia coli Nissle 1917 (EcN) naturally synthesizes and secrets heparosan as its capsular polysaccharides. In this study, we described the metabolic engineering of EcN to enhance heparosan production by optimizing the biosynthesis of precursors UDP-GlcA and UDP-GlcNAc and the expression of heparosan synthase. The orthologs of heparosan synthetic pathway enzymes from five species were expressed and comparatively investigated. bsGalU and ecKfiD for UDP-GlcA and ecGlmM for UDP-GlcNAc were introduced into EcN and the production of heparosan was increased from 0.15 g/L to 0.34 g/L, 0.39 g/L and 0.37 g/L, respectively. Combinational overexpression of bsGalU, ecKfiD and ecGlmM improved heparosan production to 0.80 g/L in flask cultures. After further upregulation of the endogenous heparosan synthases KfiAC, the titer of heparosan was improved to 1.29 g/L. Meanwhile, pathway engineering also led to the fluctuation of molecular weights between 312.39 and 410.84 kDa. Eventually, the engineered strain EC048 with overexpression of bsGalU, ecKfiD, ecGlmM and KfiAC produced 11.50 g/L heparosan in 3-L fed-batch fermentor, demonstrating EcN as a good microbial chassis is applicable for engineering an efficient heparosan cell factory.