Endocrine disturbance induces the incidence of polycystic ovary syndrome (PCOS) in women. Inhibiting adenylate cyclase (AC), which participates in the function of disturbed endocrine factors such as follicle-stimulating hormone (FSH), could be a novel therapeutic strategy to treat PCOS. In this study, a novel AC activity assay was established and used for high-throughput screening of a Food and Drug Administration (FDA)-approved drug library. Consequently, aspirin effectively inhibited the activity of AC in vitro and reduced estrogen biosynthesis by inhibiting the expression of aromatase in human granulosa cells, a key enzyme that catalyzes the conversion of androgens into estrogens. Aspirin irreversibly inhibited the activity of AC subtypes and covalently bound to recombinant AC by binding to Lys350 and Lys426, which correspond to Lys938 and Lys1014 of full-length AC2. Mechanistically, aspirin inhibited cAMP response element-binding protein-mediated aromatase expression by the AKT/mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, aspirin effectively alleviated PCOS symptoms in mouse models by decreasing androgen levels, lowering insulin levels, and reducing cystic follicles. These results revealed that aspirin is a potent inhibitor of ACs and a previously unrecognized role of ACs in estrogen biosynthesis. Therefore, aspirin warrants further investigation as a novel therapeutic agent in PCOS.© 2025 Federation of American Societies for Experimental Biology.