Difenoconazole (DCZ) is a triazole fungicide that negatively affects aquatic organisms and humans. However, data regarding the reproductive toxicity of DCZ are insufficient. In this study, we used zebrafish (from 2 h post-fertilization [hpf] to adulthood) as a model to evaluate whether DCZ at environmentally relevant concentrations (0.1, 1.0, and 10.0 μg/L) induces reproductive toxicity. After exposure to DCZ, egg production and fertilization rates were reduced by 1.0 and 10.0 μg/L. A significant decrease in gamete frequency (late vitellogenic oocytes and spermatozoa) was observed at 10.0 μg/L. The concentrations of 17β-estradiol (E2), testosterone (T), and vitellogenin (VTG) were disrupted in females and males by 1.0 and 10.0 μg/L. Exposure to 10.0 μg/L DCZ significantly inhibited the contact time between female and male fish, which was mainly achieved by affecting male fish. The transcription of genes involved in the hypothalamus-pituitary-gonad (HPG) axis was significantly changed after treatment with DCZ. Overall, these data show that the endocrine-disrupting effect of DCZ on the zebrafish HPG axis inhibited gamete maturation and disrupted reproductive behavior, reducing fertility.