Klebsiella pneumoniae synthesize large amounts of L-2,3-butanediol (L-2,3-BD), but the underlying mechanism has been unknown. In this study, we provide the first identification and characterization of an L-2,3-BD dehydrogenase from K. pneumoniae, demonstrating its reductive activities toward diacetyl and acetoin, and oxidative activity toward L-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Synthesis of L-2,3-BD was remarkably enhanced by increasing gene dosage, reaching levels that, to the best of our knowledge, are the highest achieved to date.