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Background Serum INSL3 is a Leydig cell biomarker, but little is known about the circulating concentration of INSL3 during hypothalamus-pituitary-testicular suppression. Aim To study the concomitant changes in serum concentrations of INSL3, testosterone and LH during experimental and therapeutic testicular suppression. Methods We included serum samples from three different cohorts comprising subjects before and after testicular suppression; 1) Six healthy young men who were treated with androgens (Sustanon®, Aspen Pharma, Dublin, Ireland), 2) Ten transgender girls (male sex assigned at birth) who were treated with three-monthly GnRH agonist injections (Leuprorelinacetat, Abacus Medicine, Copenhagen, Denmark) and 3) Fifty-five patients with prostate cancer who were randomized to surgical castration (bilateral subcapsular orchiectomy) or treatment with GnRH agonist (Triptorelin, Ipsen Pharma, Kista, Sweden). Serum INSL3 and testosterone concentrations were quantified in stored serum samples using validated LC-MS/MS methodologies, and LH was measured by an ultrasensitive immunoassay. Results The circulating concentrations of INSL3, testosterone and LH decreased during experimental testicular suppression in healthy young men by Sustanon injections and subsequently returned to baseline levels after release of suppression. All three hormones decreased during therapeutic hormonal hypothalamus-pituitary-testicular suppression in transgender girls and in prostate cancer patients. Conclusion INSL3 resembles testosterone as a sensitive marker of testicular suppression and reflects Leydig cell function, also during exposure to exogenous testosterone. Serum INSL3 measurements may complement testosterone as a Leydig cell marker in male reproductive disorders, during therapeutic testicular suppression as well as in surveillance of illicit use of androgens.