2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fed-batch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesis-related genes can enhance 2,3-BD production in K. pneumoniae by fermentation.