Clostridium acetobutylicum is an important industrial microorganism for n-butanol bioproduction, and its transcription factor AbrB0310 regulates various important cellular processes. However, the roles of two abrB homologues, abrB1941 and abrB3647, have not been determined because they appear inactive during transcription. Here, we performed a detailed investigation into the function of abrB1941 and abrB3647 in C. acetobutylicum. Interestingly, we observed that AbrB3647 exerts an important influence on biphasic fermentation that opposes the influence of AbrB0310, while AbrB1941 might not be essential. When abrB3647 was disrupted using the Targetron system, a greatly improved cellular growth occurred. The following analysis shows that all three AbrBs participated in metabolically regulating acidogenesis, solventogenesis, and a two-phase transition in C. acetobutylicum, but the AbrB0310 and AbrB3647 functions were the most important. Moreover, the target genes subject to AbrB0310 and AbrB3647 regulation closely overlap. Based on these results, we will better understand the roles of the three AbrBs in regulating solventogenic clostridia cell physiology.