Abstract: xylB gene inactivation can reduce xylulose phosphorylation, which leads to the reduction of xylose into xylitol without a PPP pathway. An E. coli BL21(DE3) strain with xylB gene knocked out by Red-recombination can promote the reduction yield. A pET30a-xr vector was constructed using pET30a(+) and xylose reductase gene xr from Neurospora crassa. gnd and zwf genes were amplified from genomic DNA of E. coli K-12 and cloned into pCDFDuet-1 vector to prepare pCDFDuet-gnd-zwf. The two recombined plasmids constructed were introduced into E. coli BL21(DE3). Three proteins with molecular weights of 38, 51 and 54 kD were expressed in E. coli after induced by isopropy-ß-D-thiogalactoside(IPTG). The xylose reductase specific activity of the recombinant strain is 7.25 U·mg^-1 protein, and the specific activity of 6-PGDH is 2.26 U·mg^-1 protein. Meanwhile, the specific activity of G6PDH is 1.31 U·mg^-1 protein. Fermentation results in 5 L fermenter reveal that the E. coli strain with xylB gene knocked out can promote xylitol yield. Meanwhile, the volumetric xylitol productivity of the gnd and zwf gene-transformed and xr gene containing strain is 1.04 g·(L·h)^-1, which is 24.32% higher than that of the engineered strain containing only xr gene. This result is promising for industrial application of xylitol production.