Serious concerns have been expressed about potential risks of engineered nanoparticles. Regulatory health risk assessment of such particles has become mandatory for the safe use in consumer products and medicines; also, the potential effects on reproduction and fertility are relevant for this risk evaluation. In the present study, we examined the effects of intravenously injected titanium dioxide nanoparticles (TiO2-NPs; 21 nm), with special emphasis on reproductive system. Antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase showed a significant decrease, while significant increase in lipid peroxidase was observed. Our results confirmed the bioaccumulation of TiO2-NPs in testicular cells. In TiO2-NPs-treated animals, various functional and pathological disorders, such as reduced sperm count, increase in caspase-3 (a biomarker of apoptosis), creatine kinase activity, DNA damage, and cell apoptosis were observed. Moreover, the testosterone activity was decreased significantly in a dose-dependent manner in the animals treated with TiO2-NPs as compared with control group animals. It is concluded that TiO2-NPs induce oxidative stress, which produce cytotoxic and genotoxic changes in sperms which may affect the fertilizing potential of spermatozoa.