Acetol, a C3 keto alcohol, is an important intermediate used to produce polyols and acrolein. To enhance acetol production from glycerol by Escherichia coli, a mutant (HJ02) was constructed by replacing the native glpK gene with the allele from E. coli Lin 43 and overexpression of yqhD, which encodes aldehyde oxidoreductase YqhD that converts methylglyoxal to acetol. Compared to the control strain without the glpK replacement, HJ02 had 5.5 times greater acetol production and a 53.4 % higher glycerol consumption rate. Then, glucose was added as a co-substrate to enhance NADPH availability and the ptsG gene was deleted in HJ02 (HJ04) to alleviate carbon catabolite repression, which led to a 30 % increase in the NADPH level and NADPH/NADP(+). Consequently, HJ04 accumulated up to 1.20 g/L of acetol, which is 69.0 % higher than that of HJ02. Furthermore, the gapA gene in HJ04 was silenced by antisense RNA (HJ05) to further enhance acetol production. The acetol concentration produced by HJ05 reached 1.82 g/L, which was 2.1 and 1.5 times higher than that of HJ02 and HJ04. Real-time PCR analysis indicates that glucose catabolism was rerouted from glycolysis to the oxidative pentose phosphate pathway in HJ05.