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Background: The Periploca sepium bark root (PSBR) has been regarded as a potential botanical insecticide because of its significant insecticidal activity of secondary metabolites. Several periplocosides were isolated from it as promising pesticides to control crop pests in agriculture., Results: In our research, two new periplocosides, along with four known periplocosides were isolated from PSBR. The names of new periplocosides were periplocoside T (PST) and periplocoside U (PSU) while another four periplocosides were known as follows: periplocoside A (PSA), periplocoside F (PSF), periplocoside E (PSE) and periplocoside D (PSD). All periplocosides were evalulated for insecticidal activity against 3rd Mythimna separata (Walker) and Plutella xylostella. The biometric data showed that periplocoside T, PSD and PSF had remarkable insecticidal activity against tested insects. Its values of LD 50 were 1.31, 3.94 and 3.42 μg·lavare -1 against 3rd M. separata respectively, while the activity of those compounds against 3rd P. xylostella were 5.45, 12.17 and 13.95 μg·lavare -1 , respectively. It was apparent after further study of the mechanism of action against M. separata was conducted that PST possessed the most significant insecticidal activity. The results of enzymatic activity displayed that powerful activation of tryptase, especially weak alkaline tryptase might be a dominant factor causing death of M. separata in vivo., Conclusion: We herein report isolation and the mechanisms of action of insecticidal periplocosides, which established the fundamental development of natural agents to prevent pest damage to crops. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)