Acetate is an end-product of anaerobic biodegradation and one of the major metabolites of microbial fermentation and lingo-cellulosic hydrolysate. Recently, acetate has been highlighted as a feedstock to produce value-added chemicals. This study examined acetate conversion to succinate by citrate synthase (gltA)-overexpressed Pseudomonas putida under microaerobic conditions. The acetate metabolism is initiated with the gltA enzyme, which converts acetyl-CoA to citrate. gltA-overexpressing P. putida (gltA-KT) showed an ∼50% improvement in succinate production compared to the wild type. Under the optimal pH of 7.5, the accumulation of succinate (4.73 ± 0.6 mM in 36 h) was ∼400% higher than that of the wild type. Overall, gltA overexpression alone resulted in 9.5% of the maximum theoretical yield in a minimal medium with acetate as the sole carbon source. This result shows that citrate synthase is important in acetate conversion to succinate by P. putida under microaerobic conditions.