ABC toxins are mega-Dalton bacterial pore-forming proteins and key virulence factors in bacterial pathogens of insects and some vertebrate animal species. ABC toxins have two distinct components, the binding and pore forming component, TcA, and the active component, TcBC. ABC toxins bind to the cell membrane in its pre-pore form via TcA, where, following endocytosis, acidification is hypothesised to induce pore-formation. Toxic cargo encapsulated by TcBC is then translocated through the pore formed by TcA, into the cell and elicits toxic activity. The Yersinia entomophaga toxin complex (YenTc) has a unique domain architecture that makes it a strong target for biotechnological and pharmaceutical applications. Unlike other well characterised ABC toxins, however, its physiological trigger for pore-formation is yet to be elucidated. To uncover potential mechanisms and triggers for pore-formation, we solved high resolution cryo-EM structures of YenTc in both the pre-pore and pore conformations under a variety of pH conditions designed to mimic different physiological environments. In doing so, we discovered a unique fold within YenTc that is not found in other ABC toxins characterised to date. By combining cryo-EM, biochemical assays, and molecular dynamics simulations, we found that this domain plays an important role in stabilising the pre-pore configuration of YenTc, and we discovered that this domain is proteolysed by enzymes found in the midgut of insects susceptible to YenTc. Collectively our results suggest that the action of host proteases found in the midgut of susceptible insects acts synergistically with pH changes to activate YenTc and trigger pore-formation.