[Background] D-1,2,4-butanetriol (BT) is an important four-carbon polyol with a wide range of applications in industries. The four-step biochemical reaction using xylose as a substrate is currently the most efficient BT biosynthetic route. However, the Escherichia coli host has serious carbon catabolite repression which limits the growth and BT synthesis of xylose and glucose sugar mixture. Klebsiella pneumoniae has the advantages of faster growth rate, better utilization of xylose and glucose sugar mixture. [Objective] Establishing BT synthesis pathway using xylose as a substrate in K. pneumonia, which has a weak carbon catabolite repression, to improve BT production of sugar mixture. [Methods] The heterologous pathway of BT biosynthesis was constructed in K. pneumoniae ZG25 by co-overexpressing the xylose dehydrogenase gene (xdh) from Clostridium crescenti, 2-ketoisovalerate decarboxylase gene (kivD) from Lactococcus lactis, and xylose dehydratase gene (yjhG) from E. coli W3110, obtaining recombinant strain K. pneumoniae ZG25-BT. After optimization of the medium and culture conditions and deletion of xylA, significant increase in BT production was achieved. [Results] The optimal procedure for BT production in recombinant K. pneumoniae ZG25-BT strain without the xylA was achieved in 1.5-fold LB medium under the conditions of 30.0 g/L xylose, 10.0 g/L glucose, 37 °C, 200 r/min rotation speed, adding 10.0 g/L CaCO3 to control pH and 1% inoculation amount followed by 2 h of induction with the addition of IPTG, which made BT titer, molar conversion and yield up to 4.52 g/L, 0.21 mol/mol and 15%, respectively, representing a 150%, 62% and 67% increase compared with that of unoptimizable condition, respectively. [Conclusion] A fermentative process of BT production in K. pneumoniae ZG25 is achieved. Optimization of the culture conditions and medium and the knockout of xylA are applied to improve BT production. Furthermore, this work provides a useful host for the improved BT production through metabolic engineering.