Zinc Oxide Nanoparticles (ZnO NPs) are a type of metal oxide nanoparticle with an extensive use in biomedicine. Several studies have focused on the biosafety of ZnO NPs, since their size and surface area favor entrance and accumulation in the body, which can induce toxic effects. In previous studies, ZnO NPs have been identified as a dose- and time-dependent cytotoxic inducer in testis and male germ cells. However, the consequences for the first cell stage of spermatogenesis, spermatogonia, have never been evaluated. Therefore, the aim of the present work is to evaluate in vitro the cytotoxic effects of ZnO NPs in spermatogonia cells, focusing on changes in cytoskeleton and nucleoskeleton. For that purpose, GC-1 cell line derived from mouse testes was selected as a model of spermatogenesis. These cells were treated with different doses of ZnO NPs for 6 h and 12 h. The impact of GC-1 cells exposure to ZnO NPs on cell viability, cell damage, and cytoskeleton and nucleoskeleton dynamics was assessed. Our results clearly indicate that higher concentrations of ZnO NPs have a cytotoxic effect in GC-1 cells, leading to an increase of intracellular Reactive Oxygen Species (ROS) levels, DNA damage, cytoskeleton and nucleoskeleton dynamics alterations, and consequently cell death. In conclusion, it is here reported for the first time that ZnO NPs induce cytotoxic effects, including changes in cytoskeleton and nucleoskeleton in mouse spermatogonia cells, which may compromise the progression of spermatogenesis in a time- and dose-dependent manner.