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Microbially derived, protein-based biopesticides have become a vital element in pest management strategies. Vip3 family proteins from Bacillus thuringiensis have distinct characteristics from known insecticidal Cry toxins and show efficient insecticidal activity against several detrimental lepidopteran pests. They are considered to be a promising toxic candidate for the management of various detrimental pests. In this study, we found that in addition to the preliminary digestion sites lysine, there are multiple cleavage activation sites in the linker region between domain I (DI) and DII of Vip3Aa. We further demonstrated that by adding more cleavage sites between DI and DII of Vip3Aa, its proteolysis efficiency by midgut proteases can be significantly increased, and correspondingly enhance its insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera larvae. Our study promotes the understanding of the insecticidal mechanism of Vip3 proteins and illustrates an easily implementable strategy to increase the insecticidal potency of Vip3Aa. This facilitates their potential future development and efficient application for sustainable agriculture., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Patent application on improving the insecticidal activity of Vip3A by increasing the digestion efficiency on its loop region between domain I and domain II has been filed by Shandong University., (© 2023 The Authors.)