BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility. The ketogenic diet (KD), a diet high in fat and low in carbohydrates, has been applied clinically for the treatment of obese women with PCOS. We have previously demonstrated that KD improved the reproductive phenotype in an androgen-induced PCOS mouse model, yet the underlying molecular mechanisms remain largely unclear. The aim of the present study was to investigate the effect of KD on the reproductive phenotype of a letrozole-induced PCOS mouse model. METHODS: Female C57BL/6N mice were divided into three groups, designated control, letrozole, and letrozole + KD groups. Mice of control and letrozole groups were fed the control diet, whereas letrozole + KD mice were fed a KD with 89.9% (kcal) fat for 3 weeks after the PCOS mouse model was generated. β-hydroxybutyrate (BHB), the most abundant ketone body in the body, was used to treat KGN cells with testosterone (T) to simulate the KD effect on PCOS mouse ovaries in vitro. RESULTS: Our data showed that KD treatment significantly increased blood ketone levels and reduced body weight. Ovarian functions were improved in some letrozole + KD mice. Results from in vitro experiments indicated mitochondrial damage owing to high T levels, which resulted in the leakage of cytochrome C and mitochondrial DNA into the cytosol and thus induced the activation of the intracellular caspase cascade and the cGAS-STING-NF-κB pathway, leading to granulosa cell inflammation and apoptosis. BHB exhibited certain protective effects on mitochondria of T-treated KGN cells via inhibiting the cGAS-STING pathway. Moreover, the cGAS-STING pathway was activated in ovaries of letrozole mice and was down-regulated in letrozole + KD mice. CONCLUSION: These findings, for the first time, revealed that hyperandrogenism induced ovarian dysfunction possibly through activation of the cGAS-STING pathway, which could be partially inhibited by ketone bodies produced from KD administration.