Insulin-like peptide 3 (INSL3) regulates the transabdominal phase of testicular descent in male mouse fetuses and is a biomarker of Leydig cell functionality. However, equine research remains limited. Although anti-Müllerian hormone (AMH) is a reliable diagnostic tool for equine cryptorchidism, its potential as a fertility biomarker remains unexplored. Therefore, we aimed to assess the biomarker potential of circulating INSL3, AMH, and testosterone in post-pubertal stallions. First, monthly blood samples were collected from 10 Thoroughbred stallions (age: 9.4 ± 1.6 years) for 12 months. Additionally, single-point samples were collected from 16 stallions (5-30 years) during breeding and non-breeding seasons for age-related analysis. Second, breeding (May) and non-breeding (January) samples were obtained from 25 stallions (age: 10.2 ± 4.5 years) categorized by conception rates per cycle. Serum concentrations were quantified using a time-resolved fluorescence immunoassay (for INSL3), a commercial chemiluminescent enzyme immunoassay (for AMH), and an enzyme-linked immunosorbent assay (for testosterone). The effects of circulating INSL3 and testosterone levels peaked with increasing day lengths; nadirs were observed over short day lengths. AMH lacked monthly fluctuations. However, all hormone levels decreased during non-breeding season. Age was not significantly correlated with hormone concentrations in post-pubertal breeding stallions. Contrastingly, correlations between breeding season INSL3-AMH, inter-seasonal testosterone, and AMH were observed. However, no hormonal differences were observed between fertility groups. Thus, INSL3 exhibits manipulatable seasonal dynamics, and may be useful for screening seasonal reproductivity. INSL3-AMH breeding season coordination revealed Leydig-Sertoli interplay in stallions. Nevertheless, circulating INSL3, AMH, and testosterone alone appear insufficient as fertility biomarkers in stallions.