Xylitol is a sugar–alcohol compound with broad applications in fields such as the food, dental, and pharmaceutical sectors. Although xylitol biosynthesis has gained attention, the current strategy for industrial xylitol production majorly relies on the chemical hydrogenation of xylose, which is energy-intensive and environmentally harmful. In this study, the toxicity of xylitol toward Escherichia coli was first examined, and the result demonstrated that Escherichia coli is robust against xylitol at 150 g/L. Genes encoding xylose reductases from different microorganisms were then selected and compared for xylitol production in different E. coli strains. The introduction of xylose reductase of Zymomonas mobilis, driven by the constitutive strong promoter Pgap or Pgap-6M into E. coli, resulted in the accumulation of xylitol at a titer of 64.1 g/L. The increase in NADPH by overexpressing the soluble pyridine nucleotide transhydrogenase encoded by sthA improved the xylitol titer to 83.5 g/L. Seven genes encoding xylose transporters, such as XylE and XylFGH, as well as five mutants of the xylose symporter Glf were then overexpressed and compared for xylitol production. Mutant glf^L445I exhibited the highest improvement in xylitol production at a titer of 88.4 ± 0.7 g/L and a yield of 0.95 g/g. Our study thus demonstrated that xylose reductase derived from Z. mobilis is the best one for xylitol production in E. coli, and xylitol production can be further improved by combining diverse metabolic engineering strategies. Our study, thus, provides efficient xylose reductase and a recombinant strain for future industrial xylitol production.