Perfluorooctane sulfonate (PFOS) is used extensively in industrial and household applications. High exposure to PFOS has been associated with increased odds of irregular and long menstrual cycles in women. However, the underlying mechanisms remain to be elucidated. Herein, we show that adult female mice appeared prolongation of diestrus and reduction of corpora luteum within a week of oral administration of PFOS (10 mg/kg), which are associated with decreases in the levels of serum progesterone, LH and hypothalamic GnRH. The number of AVPV-kisspeptin neurons and the AVPV-kisspeptin expression were increased in proestrus mice or OVX-mice treated with high-dose estradiol benzoate (0.05 mg/kg), which were suppressed by the administration of PFOS. The administration of PFOS or GPR54 antagonist P234 prevented the generation of LH-surge in OVX-mice treated with high-dose E2. In hypothalamic slices incubated in 100 nM E2 for 4 h, the AVPV-kisspeptin expression was significantly enhanced, which was inhibited by PFOS in a dose-dependent manner or estrogen receptor α (ERα) antagonist MPP, but not ERβ antagonist PHTPP. The incubation of ERα agonist PPT rather than ERβ agonist DPN could increase the level of AVPV-kisspeptin expression, which was sensitive to the treatment with PFOS. The administration of GPR54 agonist kisspeptin-10 in PFOS-mice could correct the prolongation of diestrus and reduction of corpora luteum, and recover the LH-surge and the levels of LH and GnRH. The results indicate that exposure to PFOS suppressed ERα-induced activation of AVPV-kisspeptin neurons leads to diestrus prolongation and ovulation reduction.