Diapause hormone (DH) is an important endocrine substance capable of influencing diapause in Lepidoptera moths that is encoded by the neuropeptide hormone DH-PBAN gene. Imidazole derivative KK-42 is a synthetic insect growth regulator that can affect diapause in Lepidoptera moths, and appears to have an opposite physiological function to DH. To test the hypothesis that KK-42 may be operating through DH to affect diapause, here, we used two Lepidoptera species Bombyx mori L. and Antheraea pernyi that enter egg and pupal diapause, respectively, through examining whether KK-42 can influence DH-PBAN and some associated mRNA expression. We found that the protein sequences of DH-PBAN in insects were highly variable, although the PRXamide C-terminus was conserved. We also found that KK-42 induced significant up-regulation and prolonged expression duration of DH-PBAN in both A. pernyi and B. mori pupae, as well as in trimolter larvae of B. mori that were induced by the application of KK-42 from the normal tetramolter larvae. In addition, KK-42 can significantly upregulate glutamic acid decarboxylase (GAD) expression in B. mori in transcriptome data. Our findings suggested that KK-42 influences diapause by upregulating GAD expression, promoting DH accumulation to prolong the secretion time of DH-PBAN.