2,6-Dichloro-1,4-benzoquinone (2,6-DCBQ), an emerging water disinfection by-product, is widely detected in water resources. However, its potential effects on the reproductive system are largely unknown. Here, we investigated the long-term effects of 2,6-DCBQ on gonadal development by exposing zebrafish from 15 to 180 days postfertilization (dpf). Following exposure to 2,6-DCBQ (20 and 100 µg/L), female-specific effects including delayed puberty onset, retarded ovarian growth and breakdown of the zona radiata were observed, resulting in subfertility in adult females. Adverse effects in folliculogenesis disappeared two months after cessation of 2,6-DCBQ administration. In contrast, no adverse impacts were noted in male testes. The effects on females were associated with significant reduction in 17β-estradiol (E2) level, suggesting a role for 2,6-DCBQ in anti-estrogenic activity. E2 level change in blood was further supported by dysregulated expression of genes (cyp19a1a, fshb, kiss3, esr2b, vtg1, and vtg3) related to the hypothalamic-pituitary-gonad-liver (HPGL) axis. The present study demonstrates for the first time that 2,6-DCBQ induces reproductive impairments in female zebrafish through disrupting 17β-estradiol level.