Introduction: Nowadays, Zinc oxide nanoparticles are considered one of the commonly used nanoparticles. They are utilized in numerous fields as agriculture, industry and biomedicine. Zinc oxide nanoparticles are widely used as food additive and in food packaging because of their antibacterial properties. Many commercial products as sun protection creams and daily-care products contain Zinc oxide nanoparticles. Aim : The aim of the study was to evaluate the subacute toxicity of different doses of orally administrated Zinc oxide nanoparticles on testis. Methods: The study was conducted on forty adult male albino rats divided into four groups (10 rats per group); Group I: control group, Group II: received 10 mg/kg/day Zinc oxide nanoparticles, Group III: received 100 mg/kg/day Zinc oxide nanoparticles, Group IV: received 200 mg/kg/day Zinc oxide nanoparticles orally for 28 days. Serum testosterone level and oxidative stress biomarkers in testicular tissue including malondialdehyde , glutathione peroxidase and superoxide dismutase were estimated. Histopathological examination of the testis by light microscope was also, performed. Results: Zinc oxide nanoparticles induced significant decrease in serum testosterone, elevation of malondialdehyde and decrease the activity of glutathione peroxidase and superoxide dismutase and glutathione peroxidase in testis in a dose dependent manner as toxicity was more obvious in high doses. Significant histopathological changes were detected. Conclusion: The study concluded that subacute oral exposure to Zinc oxide nanoparticles might cause toxic effects on testis through oxidative stress and high doses have more toxic action. Recommendations: Great attention must be given about the field of nanotoxicology to evaluate the potential toxic effects of nanoparticles on human health.