[Objective] The heat-labile enterotoxin (LT) is widely recognized as a potent mucosal immunoadjuvant. However, the mechanisms underlying its interaction with host epithelial cells remain incompletely understood, which makes it difficult to separate its toxicity from its adjuvant activity. This limitation has severely hampered its clinical application. Therefore, this study aims to elucidate the differential regulatory effects of LT and its A (LTA) and B (LTB) subunits on epithelial cells, as well as the mechanism underlying the initiation of their initial adjuvant activity. [Methods] The biologically active LTB, LTA, and its mutant LTA(R192G) were prepared via prokaryotic expression. Using the human small intestinal epithelial cell line FHs 74 Int and a mouse jejunal ex vivo intestinal segment model, we examined cell viability and apoptosis, analyzed inflammatory cytokine expression, and investigated changes in the NF-κB and NLRP3 inflammasome pathways. [Results] The three prepared proteins all possessed biological activity and could be effectively internalized by cells. LTB primarily induced early apoptosis, significantly up-regulated the expression of IL-6, IL-8, IL-1β, and TNF-α, and activated the NF-κB pathway. LTA triggered significant pyroptosis, specifically up-regulated the expression of IL-18 and IL-1β, and activated the NLRP3 inflammasome pathway. The mutant LTA(R192G) exerted weakened effects but could still activate the NLRP3 pathway. [Conclusion] The adjuvant activity of LT stems from its subunits activating immune responses in epithelial cells through different mechanisms: LTB primarily promotes pro-inflammatory cytokine production via the NF-κB pathway, while LTA mainly relies on the NLRP3 pathway to induce IL-1β/IL-18 secretion and pyroptosis. This discovery not only reveals the initial molecular events of LT-initiated mucosal immunity, but more importantly, lays a solid theoretical foundation for developing novel, safe, and efficient mucosal vaccine adjuvants through targeted modification of LTA and LTB subunits.