Serpins play a crucial role in in various physiological processes of insects. Previous studies have suggested that Serpins regulated processes like egg diapause, melanization, and antimicrobial peptide synthesis in Locusta migratoria, but their overall functional characterization remains insufficient. In this study, the functions of LmSerpin5 in regulating developmental processes and innate immunity were investigated via CRISPR/Cas9-mediated knockout. Homozygous LmSerpin5 mutation caused complete embryonic lethality. By contrast, chimeric mutants showed elevated mortality during embryonic-to-first-instar nymph transition, though chitinous tissue development remained unaffected. Additionally, adult mutants exhibited no external malformations but displayed pathological changes in immune organs, including fat body cells with enlarged lipid droplets and nuclei, and midgut absorptive cells lacking brush borders. Furthermore, pro-nymphal midguts exhibited reduced microvilli density, structural defects, and inflammatory intestinal folds. Molecular analysis confirmed upregulation of Toll pathway downstream genes (LmMyd88, LmPelle and LmTube) in mutant tissues, with midgut-specific activation of LmTube and LmPelle linking structural damage to immune dysregulation. These results demonstrated LmSerpin5 maintains homeostasis through dual mechanisms: ensuring embryonic survival and suppressing excessive Toll activation.