The 2,3-butanediol (2,3-BD) dehydrogenase gene budC of Serratia marcescens G12 was disrupted to construct the acetoin (AC) producing strain G12M. In shake-flask cultures, AC production was enhanced by increased concentrations of glucose or sodium acetate in G12M. In fed-batch fermentation, G12M produced 47.5 g/L AC along with 9.8 g/L 2,3-BD. The expression of the key enzymes for AC synthesis was further investigated. Alpha-acetolactate synthase gene budB decreased its expression significantly in G12M compared with G12. This probably explained the moderate AC production in G12M cultures. Additionally, overexpression of budB gene and α-acetolactate decarboxylase gene budA was conducted in G12M and no significant increase of AC was observed. The results suggested that intracellular AC accumulation might inhibit the expression of budB and budA gene and induce budC gene expression in G12M. Our analyses offered the bases for further genetic manipulations in improving AC production in microbial fermentations.