The Nile tilapia (Oreochromis niloticus) is economically one of the most important freshwater fish and is an excellent model for studies under laboratory conditions. Temperature is considered a very important modulator of reproductive activity in fish, although few studies have specifically addressed the effects of this key factor on morphological and functional aspects of teleost testes. Therefore, our main objectives in the present study were to analyze the effects of different temperatures (20, 25, 30, and 35°C) on testicular somatic and germ cells in sexually mature Nile tilapias. Compared with fish kept at other temperatures, tilapias maintained at 20°C demonstrated increased (P < 0.05) Sertoli cell and Leydig cell proliferation, volume density and frequency of most type B spermatogonia, and germ cell apoptosis. Conversely, tubular fluid secretion was decreased (P < 0.05) in the same animals. Although not significant, type A spermatogonia proliferation followed the pattern established for Sertoli cell and Leydig cell mitotic activity, suggesting that they preferentially would proliferate at lower temperatures. Based on most results found in our study and considering that tilapias are nonseasonal breeders, we suggest a model for temperature action on tilapia testes in which lower temperature (20°C) would favor type A spermatogonial renewal, Sertoli cell and Leydig cell proliferation, and germ cell apoptosis, whereas higher temperatures (30–35°C) would trigger rapid germ cell differentiation. Thus, tilapias could potentially be utilized in studies involving hormones and factors related to Sertoli cell and Leydig cell proliferation and spermatogonial self-renewal or differentiation.