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The 2,3-butanediol (2,3-BD) is a valuable chemical used as a solvent, liquid fuel, and precursor of many synthetic polymers and resins. Consequently, variety of researches have reported to achieve the efficient production of 2,3-BD (Zeng & Sabra, 2011) even though 2,3-BD producing level is not enough to apply industry field. Raoultella sp. B6 isolated from an oil-contaminated soil sample could produce 2,3-BD from various single carbon sources. The flask test was performed for the confirmation of 2,3-BD producing condition of the isolate such as initial pH, glucose concentration, and optimum temperature for bacterial growth and production of 2,3-BD. The pH condition and culture temperature were very critical in bacterial growth and 2,3-BD producing. Based on the batch test, fed-batch fermentation was performed to evaluate the potential 2,3-BD producing ability of the isolate under several pH condition. The initial pH 7.0 decreased to 5.5 during cell growth, and then maintained at 5.5 with 5N KOH. As a result, R. sp. B6 produced 2,3BD at highest concentration (64.7g/L), yield (0.55g/g) and productivity (0.54g/L/h). It suggests that the R. sp. B6 has potential industrial value for the 2,3-BD production through the establishment of optimum cultivative condition about variety of factors and engineering the bacterium.