We previously engineered Escherichia coli YL104 to efficiently produce succinate from glucose. In this study, we investigated the relationships between the NADH/NAD(+) ratio, ATP level, and overall yield of succinate production by using glucose as the carbon source in YL104. First, the use of sole NADH dehydrogenases increased the overall yield of succinate by 7% and substantially decreased the NADH/NAD(+) ratio. Second, the soluble fumarate reductase from Saccharomyces cerevisiae was overexpressed to manipulate the anaerobic NADH/NAD(+) ratio and ATP level. Third, another strategy for reducing the ATP level was applied by introducing ATP futile cycling for improving succinate production. Finally, a combination of these methods exerted a synergistic effect on improving the overall yield of succinate, which was 39% higher than that of the previously engineered strain YL104. The study results indicated that regulation of the NADH/NAD(+) ratio and ATP level is an efficient strategy for succinate production.