Cofactor regulation for the production of reduced metabolites, such as ethanol, plays an important role in the metabolic pathways of microorganisms. In this study, the intracellular NADH/NAD(+) ratio in the ethanologenic strain Escherichia coli KO11 was increased by the overexpression of formate dehydrogenase from Mycobacterium vaccae. To avoid the generation of pyruvate-derived byproducts, genes coding for the lactate-and acetate-producing pathways were successfully deleted using genome editing based on the CRISPR-Cas9 system. In the culture of the constructed strain, the NADH regeneration system increased the intracellular NADH/NAD(+) ratio in the presence of formate, leading to enhanced ethanol production; the amount of ethanol produced was 36% more than that produced by the original KO11 strain in culture for 24 h. A detailed investigation revealed that the transient pyruvate accumulation plays a key role in the enhanced ethanol production using the NADH regeneration system.