Despite the fact that iron represents a crucial element for the catalysis of many metabolic reactions, its accumulation in the cell leads to the production of reactive oxygen species (ROS), provoking pathological conditions such as cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, and fertility. Thus, ROS are neutralized by the enzymatic antioxidant system for the purpose of protecting cells against any damage. Iron is a potential risk factor for male fertility. However, the mechanism of action of iron on the testicular antioxidant system at the gene and protein levels is not fully understood. Thus, the purpose of the current research was to ensure a better understanding of how the long-term iron treatment influences both gene expression and enzyme activities of the testicular antioxidant system in rat testis. The data of our study showed that a significant dose-dependent increase occurred in the iron level in rat testis. A reduction occurred in reduced glutathione (GSH) levels, which represent a marker of oxidative stress, along with long-term iron overload. The expression and activity of glucose 6-phosphate dehydrogenase (G6pd), glutathione reductase (Gr), glutathione peroxidase (Gpx), and glutathione S-transferases (Gst) were significantly affected by the presence of iron. The findings of the current research demonstrate that the long-term toxic dietary iron overload influences the gene expression and enzyme activity of the testicular antioxidant defense system, but the actual effect occurs at the protein level. This may modify the sperm function and dysfunction of the male reproductive system.