This study aims to explore the use of gluconate as an alternative substrate for 2,3-butanediol (2,3-BD) production from an engineered Klebsiella oxytoca DSM 4798. Historically, glucose has been the primary substrate for microbial 2,3-BD production. However, its fermentation necessitates microaerobic conditions due to inherent redox imbalances. This study reveals that the conversion of gluconate to 2,3-BD is redox balanced, allowing for a streamlined anaerobic fermentation process, which is industrially advantageous and circumvents the late-stage 2,3-BD consumption observed in glucose microaerobic fermentation. The K. oxytoca DSM 4798 strain achieved a 2,3-BD yield of 47% and 54% from gluconate and glucose, respectively. The genetically modified MJ09 (∆ldhA ∆ackA-pta ∆frdABCD ∆adhE) strain demonstrated a significant enhancement, achieving a 2,3-BD yield of 90% from gluconate while maintaining redox balance. Meanwhile, there was no achievable growth shown for the MJ09 strain when glucose was the carbon source. This is attributed to the redox balance achieved when 2,3-BD is anaerobically produced from gluconate and it is not balanced when glucose is the carbon source. Gluconate can serve as an advantageous alternative carbon source in the production of 2,3-BD in anaerobic fermentation of K. oxytoca.