Increasing application of nanotechnology highlights the need to clarify nanotoxicity. However, few researches have focused on phytotoxicity of nanomaterials; it is unknown whether plants can uptake and transport nanoparticles. This study was to examine cell internalization and upward translocation of ZnO nanoparticles by Lolium perenne (ryegrass). The dissolution of ZnO nanoparticles and its contribution to the toxicity on ryegrass were also investigated. Zn2+ ions were used to compare and verify the root uptake and phytotoxicity of ZnO nanoparticles in a hydroponic culture system. The root uptake and phytotoxicity were visualized by light scanning electron, and transmission electron microscopies. In the presence of ZnO nanoparticles, ryegrass biomass significantly reduced, root tips shrank, and root epidermal and cortical cells highly vacuolated or collapsed. Zn2+ ion concentrations in bulk nutrient solutions with ZnO nanoparticles were lower than the toxicity threshold of Zn2+ to the ryegrass; shoot Zn contents under ZnO nanoparticle treatments were much lower than that under Zn2+ treatments. Therefore, the phytotoxicity of ZnO nanoparticles was not directly from their limited dissolution in the bulk nutrient solution or rhizosphere. ZnO nanoparticles greatly adhered on to the rootsurface. Individual ZnO nanoparticles were observed present in apoplast and protoplast of the root endodermis and stele. However, translocation factors of Zn from root to shoot remained very low under ZnO nanoparticle treatments, and were much lower than that under Zn2+ treatments, implying that little (if any) ZnO nanoparticles could translocate up in the ryegrass in this study.