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Cerium dioxide nanomaterials (CeO2 NMs) are widely used in nano-based diesel additives to decrease the emission of toxic compounds, but they have been shown to increase the emission of ultrafine particles as well as the amount of released Ce. The Organization for Economic Cooperation and Development included CeO2 NMs in the priority list of nanomaterials that require urgent evaluation, and the potential hazard of aged CeO2 NM exposure remains unexplored. Herein, human and rat sperm cells were exposed in vitro to a CeO2 NM-based diesel additive (called EnviroxTM), burned at 850 °C to mimic its release after combustion in a diesel engine. We demonstrated significant DNA damage after in vitro exposure to the lowest tested concentration (1 µg·L−1) using the alkaline comet assay (ACA). We also showed a significant increase in oxidative stress in human sperm after in vitro exposure to 1 µg·L−1 aged CeO2 NMs evaluated by the H2DCF-DA probe. Electron microscopy showed no internalization of aged CeO2 NMs in human sperm but an affinity for the head plasma membrane. The results obtained in this study provide some insight on the complex cellular mechanisms by which aged CeO2 NMs could exert in vitro biological effects on human spermatozoa and generate ROS. [ABSTRACT FROM AUTHOR]