Background and Objectives: Berberine is a yellow alkaloid, which is found in root, rhizome, skin, and stem of many plants, including barberry, turmeric, and poppy. Berberine has anti-inflammatory, antibacterial, and anti-viral properties. In the present study, the protective effect of berberine on letrozole-induced Polycystic Ovary Syndrome (PCOS), was investigated in adult female Wistar rats. Methods: In this experimental study, the rats were randomly divided into 8 groups: normal control, berberine (25, 50, and 100mg/kg intragastrically, daily) alone, PCOS control rats (letrozole, 1 mg/kg, intragastrically, daily), and rats treated with berberine (25, 50, and 100 mg/kg intragastrically, daily) together with letrozole. The administration of berberine was started 30 min before the first dose of letrozole and continued up to 28 days. The Animals were anesthetized 24 hours after the last dose of treatment. Blood sample was collected by cardiac puncture and the separated serum was evaluated to determine the levels of estrogen, progesterone, LH, FSH, and testosterone. Data were analyzed by one-way ANOVA. Results: letrozole treatment alone significantly decreased the serum levels of FSH and progesterone, while significantly increased the serum levels of LH, estrogen, and testosterone. Also, our results showed that the administration of berberine caused a significant rise in FSH, and progesterone levels and a significant decrease in the levels of LH, estrogen, and testosterone. Our results showed that treatment with berberine regulates the level of sex hormones in the letrozole-induced PCOS rats. Conclusion: The results of the present study indicated that berberine is probably useful in the treatment of letrozole-induced PCOS in rats.