<p>Cholesterol serves as an essential substrate for ovarian steroidogenesis, and its elevated levels are linked to female reproductive endocrine diseases, particularly polycystic ovary syndrome (PCOS). However, the causal role and mechanistic basis of cholesterol overload in female reproductive dysfunction remain poorly defined. In this study, we analyzed a clinical cohort of 1615 women of reproductive age and found a significant positive correlation between circulating total cholesterol and testosterone levels. To establish causality, we developed a female mouse model of hypercholesterolemia through long-term feeding with a high-cholesterol diet (HCD). Beyond hyperlipidemia and ectopic lipid deposition, these mice exhibited multiple reproductive abnormalities, including disrupted estrous cycles, elevated circulating testosterone and luteinizing hormone levels, as well as polycystic ovarian morphology, collectively reflecting key clinical characteristics of PCOS. Ovarian transcriptomic analysis in HCD-fed mice revealed aberrant activation of steroidogenesis. We further identified the transcription factor FoxO1 as a candidate regulator modulating both cholesterol- and dehydroepiandrosterone-induced ovarian steroidogenic reprogramming. Importantly, these transcriptomic changes were confirmed in granulosa cells from women with PCOS. Collectively, our findings provide direct in vivo evidence that high cholesterol contributes to PCOS pathogenesis by promoting excessive testosterone production, underscoring the necessity for lipid metabolism management to protect women's reproductive health.</p> Graphical abstract <p>Abbreviations: CL, corpus luteum; T, testosterone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; PCOS, polycystic ovary syndrome.</p>