Diflubenzuron, an insecticide commonly used in marine fish farming, has been detected in various marine environments. However, its potential impact on marine fish remains largely unknown. This study investigated the reproductive toxicity of chronic diflubenzuron exposure in female marine medaka (Oryzias melastigma). Marine medaka were exposed continuously to environmentally relevant concentrations of diflubenzuron (0.1, 1, and 10 μg/L) or a solvent control from the fertilized egg to adulthood. In exposed female marine medaka, the gonadosomatic index (GSI) and the number of laid eggs were significantly reduced. Moreover, diflubenzuron-exposed female marine medaka showed altered ovarian histopathology, with an increased relative proportion of immature oocytes and atretic follicles and a decreased relative proportion of mature oocytes. Maternal exposure to diflubenzuron also inhibited the development of the F1 generation, significantly reducing the hatching rate of F1 embryos and significantly increasing the malformation rate of F1 larvae. Furthermore, changes in hormone levels and expression of genes along the hypothalamus-pituitary-gonad-liver (HPGL) axis were observed, which may be the fundamental reason for all the reproductive toxic effects mentioned above. These results provide new insights into the impact of diflubenzuron on the female marine medaka reproductive system and underscore the importance of investigating the potential environmental risks of diflubenzuron in the marine environment.