We evaluated dehydroepiandrosterone sulfate (DHEAS) levels in premature ovarian failure (POF) patients with and without Hashimoto's thyroiditis, and the impact of DHEA supplementation on thyroid autoantibodies. In a retrospective case series, we included 67 women with spontaneous POF who received estrogen/gestagen replacement with or without DHEA (30 mg/day) for 3 months. Women who were seropositive for thyroglobulin antibodies and/or thyroperoxidase autoantibodies (n = 30) revealed lower pretherapeutic DHEAS levels (1.2 μg/ml, range 0.4-2.9 μg/ml vs. 1.9 μg/ml, range 0.2-3.9 μg/ml; p < 0.001). DHEAS showed an inverse correlation with both thyroglobulin antibodies (r = -0.426, p < 0.001) and thyroperoxidase autoantibodies (r = -0.362, p = 0.002). When treated with additional DHEA, significant decreases were found for thyroperoxidase autoantibodies (median 85.0 IU/ml, range 41-600 IU/ml vs. median 51.0 IU/ml, range 20-589 IU/ml; p = 0.005) but not for thyroglobulin antibodies.