The alteration in intracellular Zn2+ homeostasis is attributed to the generation of intracellular reactive oxygen species, which subsequently results in oxidative damage of organelles and cell apoptosis. In this work, the neurotoxic effects of ZnO hierarchical architectures (nanoparticles and microspheres, the prism-like and flower-like structures) were evaluated through the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay using RSC96 Schwann cells as the model. Cell apoptosis and cell cycle were detected using flow cytometry. The concentration of Zn2+ in the culture media was monitored using atomic absorption spectrometry. The results show that ZnO nanoparticles and microspheres displayed significant cytotoxic effects on RSC96 Schwann cells in dose- and time-dependent manners, whereas no or low cytotoxic effect was observed when the cells were treated with the prism-like and flower-like ZnO. A remarkable cell apoptosis and G2/M cell cycle arrest were observed when RSC96 Schwann cells were exposed to ZnO nanoparticles and microspheres at a dose of 80 μg/mL for 12 h. The time-dependent increase of Zn2+ concentration in the culture media suggests that the cytotoxic effects were associated with the decomposition of ZnO hierarchical architecture and the subsequent release of Zn2+. These results provide new insights into the cytotoxic effects of complex ZnO architectures, which could be prominently dominated by nanoscale building blocks.