Objective- Beta-thalassemia major (BTM) frequently results in hypogonadism, primarily due to iron overload and oxidative stress. Insulin-like growth factor-3 (INSL3), a hormone secreted by Leydig cells, may serve as a potential biomarker for the early detection of hypogonadism. This study aimed to explore the relationship between serum INSL3 levels and hypogonadism in a cohort of patients with Beta-thalassemia major.The study included 111 participants with Beta-thalassemia major, divided into three groups: 30 individuals with hypogonadism (Group I), 51 without hypogonadism (Group II), and 30 healthy controls (Group III). Hormonal and biochemical assays were performed. INSL3 was quantified using a commercial ELISA kit, while levels of testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and inhibin B were measured using chemiluminescent immunoassays on automated analyzers. Ferritin, iron, unsaturated iron-binding capacity (UIBC), and total iron-binding capacity (TIBC) were assessed using colorimetric methods on an automated biochemical analyzer. Malondialdehyde (MDA) levels were measured through the thiobarbituric acid reactive substances (TBARS) assay, and Ischemia-Modified Albumin (IMA) was determined with the albumin cobalt binding (ACB) test.Serum INSL3 levels were significantly lower in Group I compared to Groups II and III. Additionally, a notable positive correlation was observed between INSL3 and inhibin B. Multiple regression analysis identified INSL3 as an independent predictor of hypogonadism. Receiver Operating Characteristic (ROC) analysis for INSL3 demonstrated an area under the curve (AUC) of 0.893, indicating excellent diagnostic accuracy.In patients with Beta-thalassemia major, INSL3 levels are diminished in the presence of hypogonadism, alongside elevated markers of oxidative stress and increased iron overload. This finding highlights the potential connection between INSL3 and male hypogonadism in the context of BTM.