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Androstenedione (AED) is a naturally occurring androgen that is released into the environment through human and animal excretion. Several studies have shown that AED can interfere with the normal growth, development, and reproduction of organisms. However, studies examining the thyroid system, an important part of endocrine system of fish, are still lacking. This study examines thyroid hormone levels including T3 and T4, and transcriptional expression levels of genes associated with the hypothalamic-pituitary-thyroid axis (HPT axis) in adult zebrafish. Fish were exposed to a solvent control and three other concentrations of AED (0.2, 2.3, and 23.7 μg/L) for 60 days. Results indicated that AED significantly increased T4 levels but inhibited transcription of some HPT axis-related genes (nis, tpo, tg, dio1, dio2, sult1 st5, pax8, and nkx2.1) in female zebrafish. The adverse effects of AED on the thyroid system in males were very low, only affecting down-regulation of the tshb gene and up-regulation of the thrb gene. The overall results demonstrated that AED interferes with the thyroid endocrine system in fish.