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BACKGROUND: Myzus persicae (Hemiptera: Aphididae) is one of the most notorious pests of many crops worldwide. Most Cry toxins produced by Bacillus thuringiensis show very low toxicity to M. persicae; however, a study showed that Cry41‐related toxin had moderate toxic activity against M. persicae. In our previous work, potential Cry41‐related toxin‐binding proteins in M. persicae were identified, including cathepsin B, calcium‐transporting ATPase, and Buchnera‐derived ATP‐dependent 6‐phosphofructokinase (PFKA). Buchnera is an endosymbiont present in almost all aphids and it provides necessary nutrients for aphid growth. This study investigated the role of Buchnera‐derived PFKA in Cry41‐related toxicity against M. persicae. RESULTS: In this study, recombinant PFKA was expressed and purified, and in vitro assays revealed that PFKA bound to Cry41‐related toxin, and Cry41‐related toxin at 25 μg ml−1 significantly inhibited the activity of PFKA. In addition, when M. persicae was treated with 30 μg ml−1 of Cry41‐related toxin for 24 h, the expression of dnak, a single‐copy gene in Buchnera, was significantly decreased, indicating a decrease in the number of Buchnera. CONCLUSION: Our results suggest that Cry41‐related toxin interacts with Buchnera‐derived PFKA to inhibit its enzymatic activity and likely impair cell viability, resulting in a decrease in the number of Buchnera, and finally leading to M. persicae death. These findings open up new perspectives in our understanding of the mode of action of Cry toxins and are useful in helping improve Cry toxicity for aphid control. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]