Butanol biosynthesis through aldehyde/alcohol dehydrogenase (adhE2) is usually limited by NADH availability, resulting in low butanol titer, yield, and productivity. To alleviate this limitation and improve n-butanol production by Clostridium tyrobutyricum ack-adhE2 overexpressing adhE2, the NADH availability was increased by using methyl viologen (MV) as an artificial electron carrier to divert electrons from ferredoxin normally used for H-2 production. In the batch fermentation with the addition of 500M MV, H-2, acetate, and butyrate production was reduced by more than 80-90%, while butanol production increased more than 40% to 14.5g/L. Metabolic flux analysis revealed that butanol production increased in the fermentation with MV because of increased NADH availability as a result of reduced H-2 production. Furthermore, continuous butanol production of approximate to 55g/L with a high yield of approximate to 0.33g/g glucose and extremely low ethanol, acetate, and butyrate production was obtained in fed-batch fermentation with gas stripping for in situ butanol recovery. This study demonstrated a stable and reliable process for high-yield and high-titer n-butanol production by metabolically engineered C. tyrobutyricum by applying MV as an electron carrier to increase butanol biosynthesis. Biotechnol. Bioeng. 2015;112: 705-715. (c) 2014 Wiley Periodicals, Inc.