Abstract <p>This study identified thrombospondin-1 (THBS1) as a potential biomarker for polycystic ovary syndrome (PCOS) and its pivotal role in disease pathogenesis through multi-level bioinformatics analysis and experimental validation. By integrating five PCOS-related datasets and applying multiple machine learning techniques, THBS1 was identified as the most specific differentially expressed gene. Clinical sample analysis confirmed significantly elevated THBS1 levels in PCOS patients (p &lt; 0.0001). In DHEA-induced PCOS cell and animal models, THBS1 upregulation was accompanied by marked suppression of PI3K/AKT signaling pathway activity. THBS1 knockdown significantly restored PI3K/AKT pathway activity in PCOS cells, indicating THBS1 participates in PCOS pathogenesis by negatively regulating this pathway. Animal experiments corroborated in vitro findings, revealing increased THBS1 expression and reduced PI3K/AKT pathway activity in ovarian tissues of PCOS model rats. Molecular docking studies predicted strong binding affinity between pioglitazone hydrochloride and THBS1, suggesting novel therapeutic approaches. This study systematically elucidates for the first time the pivotal role of the THBS1/PI3K/AKT axis in PCOS pathogenesis, providing novel molecular targets and a theoretical foundation for precision diagnosis and personalized treatment.</p> New and noteworthy <p>This study systematically demonstrates for the first time the value of THBS1 as a potential biomarker for PCOS. Through multi-level analysis, it confirms that THBS1 participates in the pathogenesis of PCOS by negatively regulating the PI3K/AKT signaling pathway, providing new molecular targets and a theoretical foundation for precise diagnosis and personalized treatment.</p>