[Objective] In order to improve the D-1,2,4-butanetriol (BT) titer in recombinant Escherichia coli, two by-product pathways were blocked. [Methods] The xylAB, yagE and yjhH were knocked out by Red system and the cell growth, BT production and accumulation of byproduct of the resultant strains were detected. [Results] The biomass and BT titer of xylAB-deficient strain were repressed by 57% and 20%, with the BT yield per cell increased by 84%. In contrast, the biomass of yagE-deficient and yjhH-deficient strains was increased by 10% and 5%, respectively. The BT titers of these two strains showed increase of 36% and 14%, respectively. Co-knocking out these two genes led to the decrease of biomass by 21%, but the BT titer was improved by 184%, up to 2.44 g/L, and BT yield per cell was increased by 258%. Meanwhile, blocking these two by-product pathways simultaneously resulted in the decrease of biomass by 72% and raised BT titer per cell by 4 times, and the final BT titer was showed 43% improvement. The xylonate titers of recombinant strains were decreased, and the BT titer was further improved by pH-controlled fermentation, up to 3.11 g/L. [Conclusion] Although knocking out xylAB is beneficial to BT produce, the repressed xylose utilization ability through the PPP pathway and accumulated xylonate lead to the reduced biomass and BT titer. Knocking out the yagE or yjhH showed slightly positive effect on BT production. Further co-deficiency of these two genes shifts more 2-keto-3-deoxy-xylonate (KDX) into BT pathway, leading to significantly improved BT titer. Finally, blocking these two by-product pathways shows negative effect on cell growth but slightly improvement on BT production.