Excessive consumption of fluoride (F) through drinking, eating, and/or environmental contaminants induces chronic toxicity known as fluorosis. Our previous research has shown that fluorosis was associated with male reproductive disorders. The current study is designed to explain the protective effect of vitamin E (VE), insulin-like growth factor-I (IGF-I), and human chorionic gonadotropin (hCG) against natrium fluoride (NaF)-induced alterations in isolated Leydig cells (LCs). These NaF-induced alterations include decreased cell proliferation, steroidogenesis, and relative gene expression. Isolated LCs were incubated with NaF (0, 5, 20 mg/L) and/or 10 ?g/ml VE, 100 ng/ml IGF-I, and 100 IU/ml hCG. NaF-treated cells’ ability to secrete testosterone (T) was significantly less than other treated groups (P < 0.05). Additionally, in NaF-treated cells, there was a significant upregulation of certain relative mRNA expressions such as Star and Cyp11a, as well as significantly less cell proliferation in a dose-dependent manner (P < 0.05). These data clearly show that VE, IGF-1, and hCG have a protective effect in the LCs functions. Taken together, the final results of this study shown herein are consistent with the assumption that VE, IGF-I, and hCG volunteered ameliorative effects against the deleterious effects of NaF through their protective activity. Although it is hypothesized that ameliorative effects might have been involved, the fundamental pathway(s) remain(s) to be illuminated.