Sunset Yellow (SY) (E110) is a synthetic yellow azo dye commonly found in various food products such as orange sodas, powdered marinades, snack chips, ice creams, apricot jam, gelatin desserts, and cake decorations. On the other hand, Diethylnitrosamine (DEN) belongs to the nitrosamine family and is used in various applications such as a gasoline additive, lubricant additive, stabilizer in plastics, solvent in the fiber industry, and is even present in tobacco smoke. The objective of this study was to investigate the effects of SY and DEN on the testes of rats. Twenty-four male Sprague Dawley (SD) rats, aged eight weeks, were divided into four groups. The first group served as the normal control and received no treatment. The second group was solely treated with SY at a dosage of 160 mg/kg/b.wt./day via intragastric gavage (ig). The third group received an intraperitoneal (IP) injection of DEN at a dosage of 200 mg/kg. The fourth group underwent the same DEN injection as the second group, followed by treatment with SY at a dosage of 160 mg/kg/b.wt./day administered via intragastric gavage (ig). The results of the study revealed that the testes weight increased in the treatment groups (3.3±0.63; 2.95±0.63 and 3.32±0.64, for SY, DEN, and the combination of SY and DEN, respectively) compared to the control group (2.9±0.31). In terms of the lipid profile, the DEN group exhibited increased levels of serum cholesterol, triglycerides, and low-density lipoprotein (LDL), accompanied by a decrease in high-density lipoprotein (HDL). Conversely, the group treated with SY demonstrated an increase in cholesterol, triglycerides, and LDL levels, but a decrease in HDL levels. Additionally, the combination of SY and DEN showed a reduction in Glutathione reductase and Glutathione peroxidase, which are antioxidant enzymes. Furthermore, the DEN group displayed significant microscopic shrinkage of seminiferous tubules, desquamation of germinal epithelium, and increased interstitial tissue with haemorrhage and haemolysis. In contrast, the group treated with SY showed mild degenerative changes in seminiferous tubules, highly congested blood vessels, and interstitial haemorrhage.