Periplocosides, extracted from the root bark of Periploca sepium, are plant secondary compounds known to inhibit the V-ATPase enzyme in susceptible insect species, such as Mythimna separata. However, many species, including Spodoptera exigua, show resistance to these compounds. Previous studies identified the V-ATPase subunit A (VATP-A) in the midgut epithelium of M. separata as the putative target of periplocoside P (PSP), but the specific amino acids involved in this interaction remained unclear. In this study, we demonstrate the selective toxicity of PSP and its inhibition effect on V-ATPase. Molecular docking identified potential interactions between PSP and three amino acids (K85, R171, E199) in MsVATP-A, with in vitro binding assays revealing that K85 and R171 serve as the primary binding sites. Notably, sequence alignment revealed that R171 in sensitive species is substituted with K in resistant species. To investigate the functional implications of this substitution, we performed in vitro site-directed mutagenesis to exchange the corresponding amino acids between the VATP-A orthologs of M. separata and S. exigua. The R171K mutation in MsVATP-A reduced binding to PSP, while the K170R mutation in SeVATP-A enhanced it. Furthermore, in vivo genome editing in Drosophila melanogaster, a PSP-sensitive species, revealed that the R171K mutation conferred 15.78-fold resistance to PSP compared to the wild-type strain (w