Heparosan is the start point for chemoenzymatic synthesis of heparin and it is of great significance to efficiently synthesize heparosan in microorganisms. The effects of overexpressing key enzyme genes of the UDP-glucuronic acid (UDP-GlcUA) pathway (pgcA, gtaB and tuaD) or the UDP-N-acetyl-glucosamine (UDP-GlcNAc) pathway (glmS, glmM and glmU) on the heparosan production and molecular mass were analyzed in the constructed heparosan-producing Bacillus subtilis ((1.71±0.08) g/L). On this basis, heparosan production was increased to (2.89±0.11) g/L with the molecular mass of (75.90±1.18) kDa through co-overexpressing the tuaD, gtaB, glmU, glmM and glmS genes in shake flask cultivation. In the 3 L fed-batch fermentation, heparosan production was improved to (7.25±0.36) g/L with the molecular mass of (46.66±2.71) kDa, providing the potential for heparosan industrial production.