Clostridia produce autoinducing peptides (AIPs) regulated by the accessory gene regulator (Agr) quorum sensing system, playing a critical role in intercellular communication. However, the biosynthetic pathway and regulatory functions of clostridial AIPs remain inadequately characterized. In this study, we employed chemical quantification, genetic investigations, and in vitro reconstitution experiments to elucidate the native Ca-AIP in Clostridium acetobutylicum, a prominent industrial producer of acetone, butanol, and ethanol. Our findings identified a signal peptidase (Cac1760) and two CAAX metalloproteases (Cac0077 and Cac2478) as key players in N-terminal cleavage, while AgrB was found to be essential for C-terminal processing during Ca-AIP biosynthesis. Notably, overexpression of agrBD led to a 4.4-fold enhancement in Ca-AIP formation, which corresponded with an increase in butanol production from 12.5 to 14.9 g/L, while preserving vegetative cell morphology. The direct involvement of Ca-AIP in both butanol production and maintenance of cell morphology was further validated through exogenous supplementation. Collectively, these results provide novel insights into the biosynthesis of AIPs and propose a promising strategy for optimizing microbial processes in industrial applications.