Klebsiella oxytoca mutants were isolated from the wild type strain ME-303 after mutagenesis with UV coupled with diethyl sulfate and the following modified proton suicide method. By analyzing the activities of lactate dehydrogenase and phosphotransacetylase involved in lactic and acetic acid formation pathways and batch fermentation, one mutant, ME-UD-3, was isolated that produced 7.8% more 2,3-butanediol than ME-303 with the corresponding byproducts of lactic and acetic acid decreased by 88% and 92%, respectively.