Because androgen function is regulated by its receptors, androgen-androgen receptor signaling is crucial for regulating spermatogenesis. Androgen is mainly testosterone secreted by testis. Based on the results of early studies in goats, the administration of melatonin over an extended period of time increases steroid production, but the underlying mechanism remains unclear. Here, we report the expression of the melatonin membrane receptors MT1 and MT2 and the retinoic acid receptor-related orphan receptor-alpha (RORα) in the goat testis. An in vitro differentiation system using spermatogonial stem cells (SSCs) cultured in the presence of testicular somatic cells was able to support the formation of sperm-like cells with a single flagellum. The addition of 10^-7 M melatonin to the in vitro culture system increased RORα expression and considerably improved the efficiency of haploid cell differentiation, and the addition of the RORα agonist CGP52608 significantly increased the testosterone concentration and expression of GATA binding factor 4 (GATA-4). Furthermore, inhibitors of melatonin membrane receptors and a RORα antagonist (T0901317) also led to a considerable reduction in the efficiency of haploid spermatid formation, which was coupled with the suppression of GATA-4 expression. Based on these results, RORα may play a crucial role in enhancing melatonin-regulated GATA-4 transcription and steroid hormone synthesis in the goat spermatogonial stem cell differentiation culture system.