BACKGROUNDPyriproxyfen is an insect growth regulator (IGR) that is effective against various types of insect pests. However, the molecular mechanism underlying pyriproxyfen effects on insect reproduction remains unclear. Thus, in this study, we attempted to uncover the mechanisms underlying the impact of pyriproxyfen on the reproductive system of the model organism Drosophila melanogaster.RESULTSA significant decrease in Drosophila reproduction was observed after pyriproxyfen treatment. The juvenile hormone (JH) titer was significantly increased (120.4%) in the ovary samples of pyriproxyfen‐treated flies. Likewise, the concentrations of key enzymes and the expression of key genes related to the JH signaling pathway were also increased in the pyriproxyfen‐treated group compared with the control group. Furthermore, pyriproxyfen treatment significantly increased (15.6%) the number of germline stem cells (GSCs) and significantly decreased (17%) the number of cystoblasts (CBs). However, no significant differences were observed in the number of somatic cells. We performed RNA interference (RNAi) on five key genes (Met, Tai, gce, ftz‐f1, and hairy) related to the JH signaling pathway in germ cells using the germ cell‐specific Gal4 driver. Interestingly, RNAi of the selected genes significantly decreased the number of both GSCs and CBs in pyriproxyfen‐treated transgenic flies. These results further validate that pyriproxyfen enhances GSC proliferation by up‐regulating JH signaling.CONCLUSIONOur results indicate that pyriproxyfen significantly decreases reproduction by affecting germ cells in female adult ovaries. The effect of pyriproxyfen on germ cell proliferation and differentiation is mediated by an increase in JH signaling. This study has significant implications for optimizing pest control strategies, developing sustainable agriculture practices, and understanding the mechanism of insecticide action. © 2024 Society of Chemical Industry.