Abstract: This study aims at overexpressingBudC and then upregulating the synthesis of 2,3-butanediol (2,3- BD). We constructed a recombinant overexpression vector named pR1SW-BudC by genetic engineering technology, after the PCR and enzyme digestion identification, pR1SW-BudC was transformed intoKlebsiella oxytoca HD79 by electric conversion, K. oxytoca HD79- 01 was obtained by kanamycin screening. Using original strain as the control, 2,3-BD content and the relative enzyme activity was detected with shaking flask fermentation. Furthermore, expression difference of BudC was detected with qRT- PCR. Finally, BudC expression in HD79-01 was 45.25 times as much as that of HD79 (P <0.01). The shaking flask fermentation showed that the maximum of yield, conversion rate and production strength increased by 24.54%, 10.97% and 45.08%, respectively (30.818 ± 0.003 g/L, 0.253 ± 0.013 g/g, 0.43 ± 0.01 g/(L·h)). The enzyme activity increased by 3.61 times (0.563 ± 0.003 U/mg). Thus, the construction ofBudC overexpressK. oxytoca HD79 strain not only increased 2,3-BD yield, but also laid the foundation for the industrial production of 2,3-BD.