Abstract: Acetic acid is one of the major metabolic by-products in 2,3-butanediol classical metabolic pathway. Production of 2,3-butanediol can be affected by synthesis of numerous acetic acid which will lead to changing of environmental pH and inhibition of cell growth. Acetate kinase is a key enzyme in acetic acid metabolic pathway, knock-out of encoding genes of acetate kinase will theoretically decrease and even remove the production of acetic acid, thus the yield and productivity of 2,3-butanediol will be enhanced. In this paper, 856 bp of partial kinase gene ack without base deletion was cloned taking genomic DNA of Klebsiella oxytoca and plasmid pT-ack as templates with primers designed according to similarity of ack sequences from Klebisella oxytoca KCTC1686. The ack gene was located in open reading frame of ack gene of Klebisella oxytoca KCTC1686 though integrated ORF was not found in it by ORF analysis. The result demonstrated that the ack gene was partially acetate kinase gene from Klebisella oxytoca HD79 which laid the foundation for further construction of acetate kinase mutant of Klebisella oxytoca HD79 and provided reference for other relative researches.