Because of their insolubility in water, nanoparticles have a limitation concerning toxicity experiments. The present study demonstrated a plant agar test for homogeneous exposure of nanoparticles to plant species. The effect of Cu nanoparticles on the growth of a plant seedling was studied, and bioaccumulation of nanoparticles was investigated. All tests were conducted in plant agar media to prevent precipitation of water-insoluble nanoparticles in test units. The plant species were Phaseolus radiatus (mung bean) and Triticum aestivum (wheat). Growth inhibition of a seedling exposed to different concentrations of Cu nanoparticles was examined. Copper nanoparticles were toxic to both plants and also were bioavailable. The 2-d median effective concentrations for P. radiatus and T. aestivum exposed to Cu nanoparticles were 335 (95% confidence level, 251–447) and 570 (450–722) mg/L, respectively. Phaseolus radiatus was more sensitive than T. aestivum to Cu nanoparticles. A cupric ion released from Cu nanoparticles had negligible effects in the concentration ranges of the present study, and the apparent toxicity clearly resulted from Cu nanoparticles. Bioaccumulation increased with increasing concentration of Cu nanoparticles, and agglomeration of particles was observed in the cells using transmission-electron microscopy–energy-dispersive spectroscopy. The present study demonstrated that the plant agar test was a good protocol for testing the phytotoxicity of nanoparticles, which are hardly water soluble.