The cell permeability and cytotoxic effects of different-sized zinc oxide (ZnO) particles were investigated using a human colorectal adenocarcinoma cell line called Caco-2. Morphological observation by scanning electron microscopy revealed that three zinc oxides with different mean particle sizes (ZnO-1, 20 nm; ZnO-2, 90~200 nm; ZnO-3, ) tended to aggregate, particularly in the case of ZnO-1. When cytotoxicities of all three sizes of zinc oxide particles were measured at concentration ranges of /mL, significant decreases in cell viability were observed at concentrations of /mL and higher. Among the three zinc oxides, ZnO-1 showed the lowest viability at /mL in Caco-2 cells, followed by ZnO-2 and ZnO-3. The permeate concentration of ZnO-1 from the apical to the basolateral side in the Caco-2 model system after four hours was about three-fold higher than that of either ZnO-2 or ZnO-3. These results demonstrated that ZnO-1, with a 20 nm mean particle size, had poorer viability and better permeability in Caco-2 cells than ZnO-2 and ZnO-3.