The innate immune system of insects include the Toll pathway, which is mediated by an extracellular serine proteinase cascade. In the tobacco hornworm, Manduca sexta, hemolymph proteinase 8 (HP8) promotes the synthesis of antimicrobial proteins by cleaving proSpätzle, the putative ligand of M. sexta Toll. HP8 has been observed to form a complex in hemolymph with M. sexta serpin-1, which has multiple alternative splicing isoforms. To investigate the regulation of HP8 and its processing of proSpätzle, we characterized the interaction of recombinant HP8 with serpin-1 isoform J (serpin-1J). Recombinant serpin-1J formed an SDS-stable complex with HP8 in vitro. The association rate constant of serpin-1J and HP8 was 1.3 × 10 4 M −1 s −1, with a stoichiometry of inhibition of 5.4. Serpin-1J inhibited the cleavage of proSpätzle by HP8. Injection of serpin-1J into M. sexta larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced expression of cecropin, attacin and hemolin mRNA in fat body. Altogether, these results suggest that serpin-1J functions to inhibit HP8 and thereby modulates the concentration of active Spätzle to regulate the Toll pathway response in M. sexta.