Smoothened (SMO) is a seven-transmembrane protein related to the G protein-coupled receptor and plays a central role as transducer in the Hedgehog (Hh) signaling pathway, which has been demonstrated to play an important role in the differentiation and development of gonadal cells in mammals. However, the role of Smo signaling in fish testis development remains unknown by now. To elucidate the role of Smo signaling in Oreochromis niloticus testis, the O. niloticus smo (Onsmo) cDNA was cloned and characterized. Its expression profile in different tissues was investigated by RT-PCR and in testis by fluorescence in situ hybridization (FISH). After treatment with Smo specific agonist SAG and inhibitor cyclopamine, the proliferation and apoptosis of germ cells and somatic cells in an ex vivo testicular organ culture system were evaluated by EdU incorporation and TUNEL staining, respectively. The full open reading frame of Onsmo is 2 478 bp in length, it encodes a putative 825 amino acids protein, including seven transmembrane domains. The identity of amino acid sequence with human SMO is up to 77%. Onsmo was expressed in multiple examined tissues including testis. According to FISH results, Onsmo in testis was expressed in spermatogonia, spermatocytes, Sertoli cells (SC) and Leydig cells (LC). In the testicular organ culture system, our experimental data show that SAG significantly promoted the proliferation of spermatogonia, whilst cyclopamine significantly promoted the apoptosis of testicular SC and LC. Taken together, our study suggests that Smo signaling plays an important role in the proliferation of spermatogonia and the survival of testicular somatic cells in O. niloticus. To our best knowledge, this study firstly reports the role of Smo signaling in fish testicular cells. This study not only enriches our understanding of the role of Smo signaling in fish testicular cells, but also lays an important foundation for further elucidation of its mechanism.