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Radiation-induced sterile insect technique is a biologically based, environment-friendly method for the suppression or eradication of a number of insect pests. Although the basic mechanisms underlying the technology have been well studied, little is known about the cell responses in organisms. Characterization of the metabolic shift associated with radiation exposure in sterile insects would be helpful for understanding the detailed mechanism underlying this technique and promote its practical application. In this article, a metabolomic study was performed to characterize the global metabolic changes induced by radiation using untreated and 40 Gy 60Coγ-irradiated testes of Japanese pine sawyer, Monochamus alternatus Hope. Differential metabolites were detected and tentatively identified. Many key metabolites in glycolysis and the tricarboxylic acid cycle, as well as most fatty and amino acids, were elevated in irradiated male M. alternatus, presumably resulting from depression of glycolysis and the tricarboxylic acid cycle, each of which are important pathways for energy generation Adenosine Triphosphate (ATP) in insect spermatozoa. The findings in this article will contribute to our knowledge of the characteristic metabolic changes associated with irradiation sterility and understand the molecular mechanisms underlying radiation-induced sterile insect technique.