Due to their beneficial properties, the use of zinc oxide nanoparticles (ZnO NP) is constantly increasing, especially in consumer-related areas, such as food packaging and food additives, which is leading to an increased oral uptake of ZnO NP. Consequently, the aim of our study was to investigate the cellular uptake of two differently sized ZnO NP (<50 nm and <100 nm; 12-1229 µmol/L) using two human intestinal cell lines (Caco-2 and LT97) and to examine the possible resulting toxic effects. ZnO NP (<50 nm and <100 nm) were internalized by both cell lines and led to intracellular changes. Both ZnO NP caused time- and dose-dependent cytotoxic effects, especially at concentrations of 614 µmol/L and 1229 µmol/L, which was associated with an increased rate of apoptotic and dead cells. ZnO NP < 100 nm altered the cell cycle of LT97 cells but not that of Caco-2 cells. ZnO NP < 50 nm led to the formation of micronuclei in LT97 cells. The Ames test revealed no mutagenicity for both ZnO NP. Our results indicate the potential toxicity of ZnO NP after oral exposure, which should be considered before application.