Bacillus subtilisis a preferred microbial host for the industrial production of nutraceuticals and a promising candidate for the synthesis of functional sugars, such asN-acetylglucosamine (GlcNAc). Previously, a GlcNAc-overproducerB. subtilisSFMI was constructed usingglmSribozyme dual-regulatory tool. Herein, we further engineered to enhance carbon flux from glucose towards GlcNAc synthesis. As a result, the increased flux towards GlcNAc synthesis triggered phosphosugar stress response, which caused abnormal cell growth. Unfortunately, the mechanism of phosphosugar stress response had not been elucidated inB. subtilis. To reveal the stress mechanism and overcome its negative effect in bioproduction, we performed comparative transcriptome analysis. The results indicate that cells slow glucose utilization by repression of glucose import and accelerate catabolic reactions of phosphosugar. To verify these results, we overexpressed the phosphatase YwpJ, which relieved phosphosugar stress and allowed us to identify the enzyme responsible for GlcNAc synthesis from GlcNAc 6-phosphate. In addition, the deletion ofnagBBandmurQ, responsible for GlcNAc precursor degradation, further improved GlcNAc synthesis. The best engineered strain,B. subtilisFMIP34, increased GlcNAc titer from 11.5 to 26.1 g/L in shake flasks and produced 87.5 g/L GlcNAc in 30-L fed-batch bioreactor. Our results not only elucidate, for the first time, the phosphosugar stress response mechanism inB. subtilis, but also demonstrate how the combination of rational metabolic engineering with novel insights into physiology and metabolism allows the construction of highly efficient microbial cell factories for the production of high-value chemicals.