Polycystic ovarian syndrome (PCOS), the most common endocrine disorder of women in reproductive age, is typical with hyperandrogenism and disturbance of the hypothalamus–pituitary–ovary (HPO) axis, i.e. abnormal expression of hypothalamic gonadotropin-releasing hormone (GnRH) followed by the elevated ratio of serum luteinizing hormone (LH) level to follicle-stimulating hormone (FSH) level. This derangement might have a close relationship with hypothalamic kisspeptin expression that is thought to be a key regulator of GnRH. Crocetin, one of the main components of Saffron clinically used as traditional medicine in gynecology diseases, was evaluated for its therapeutic effects on PCOS induced by prenatally exposure to dihydrotestosterone (DHT) in mice. Herein, we found that DHT-treated mice showed a similar phenotype to human PCOS such as heavier ovary, prolonged diestrus, multiple enlarged follicles with fewer corpus luteum, and higher LH and testosterone levels. Kisspeptin expression was lower in anteroventral periventricular nucleus (AVPV) but higher in arcuate nucleus (ARC). Treatment of crocetin prevented the prolongation of diestrus and reduction in corpora luteum, recover the levels of GnRH, FSH, LH, progesterone (P4), estradiol (E2) and testosterone (T), and increase the kisspeptin level in AVPV but reduce that in ARC. The present study provides in vivo evidence that crocetin improved the PCOS in mice via increasing AVPV-kisspeptin and reducing ARC-kisspeptin expression.