Increased production and use of zinc oxide nanoparticles (ZnO-NPs) in consumer products has prompted the scientific community to investigate their potential toxicity, and understand their impact on the environment and organisms. Molecular mechanisms involved in ZnO-NP toxicity are still under debate and focus essentially on high dose expositions. In our study, we chose to evaluate the effect of sub-toxic doses of ZnO-NPs on human hepatocytes (HepG2) with a focus on metal homeostasis and redox balance disruptions. We showed massive dissolution of ZnO-NPs outside the cell, transport and accumulation of zinc ions inside the cell but no evidence of nanoparticle entry, even when analysed by high resolution TEM microscopy coupled with EDX. Gene expression analysis highlighted zinc homeostasis disruptions as shown by metallothionein 1X and zinc transporter 1 and 2 (ZnT1, ZnT2) over-expression. Major oxidative stress response genes, such as superoxide dismutase 1, 2 and catalase were not induced. Phase 2 enzymes in term of antioxidant response, such as heme oxygenase 1 (HMOX1) and the regulating subunit of the glutamate-cysteine ligase (GCLM) were slightly upregulated, but these observations may be linked solely to metal homeostasis disruptions, as these actors are involved in both metal and ROS responses. Finally, we observed abnormal mitochondria morphologies and autophagy vesicles in response to ZnO-NPs, indicating a potential role of mitochondria in storing and protecting cells from zinc excess but ultimately causing cell death at higher doses.