Anti-Mullerian hormone (amh) is one of the earliest functional genes expressed during testicular differentiation. It has been suggested that androgen signaling regulates critical genes for the differentiation and development of the testis. To elucidate the exact regulatory mechanisms involved in amh transcription mediated by androgen signaling, androgen signaling was manipulated in zebrafish by cytochrome P450 17a1 (cyp17a1) knockout and Flutamide treatment. In cyp17a1-deficient and Flutamide-treated testes, up-regulated sry-box9a (sox9a) and down-regulated amh were observed. Moreover, a physical association of the zebrafish androgen receptor (AR) and SOX9A was found. The interaction between AR and SOX9A was mediated via the DNA binding domain (DBD) of AR and the transactivation domain (TA) of SOX9A, and was further enhanced by 5-alpha dihydrotestosterone (DHT), one of the most potent androgens. Intriguingly, together with SOX9A, androgen signaling synergistically promoted amh transcription, mainly through the proximal 1 kb of the amh promoter region. Taken together, our data demonstrate a critical mechanism underlying the direct synergy of androgen signaling and SOX9A in the regulation of amh transcription. (C) 2017 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.