To improve the production and molecular mass of the glycosaminoglycan hyaluronan (HA) in Bacillus subtilis by engineering hyaluronan synthase (HAS) from Streptococcus zooepidemicus. By mutating regions within HAS intracellular domains, five positive variants exhibiting higher HA production (from 1.22 to 2.24 g l(-1)) and molecular mass values (from 1.20 to 1.36 x 10(6) Da) were constructed and characterized. Overexpression of the V5 variant and the genes tuaD and glmU increased HA production and molecular mass to 2.8 g l(-1) and 2.4 x 10(6) Da, respectively. This study provides a novel strategy for improving HA production and its molecular mass.