Objective To improve enzyme activity of Glucosamine-6-phosphate synthase (Glms) ofBacillus subtilisby site saturation mutagenesis at Leu(593), Ala(594), Lys(595), Ser(596)and Val(597)based on computer-aided semi-rational design. Results The results indicated that L593S had the greatest effect on the activity of BsGlms and the enzyme activity increased from 5 to 48 U/mL. The mutation of L593S increased the yield of glucosamine by 1.6 times that of the original strain. The binding energy of the mutant with substrate was reduced from - 743.864 to - 768.246 kcal/mol. Molecular dynamics simulation results showed that Ser(593)enhanced the flexibility of the protein, which ultimately led to increased enzyme activity. Conclusion We successfully improved BsGlms activity through computer simulation and site saturation mutagenesis. This combination of methodologies may fit into an efficient workflow for improving Glms and other proteins activity.