Internalization and phytotoxic effects of CuO nanoparticles (nCuO) in plants were studied at the cellular level. Arabidopsis thaliana was hydroponically challenged by nCuO (100 mg/L), as compared to Cu2+ ions (1.2 mg/L), to account for nCuO dissolution for 96 h and 28 days to monitor Cu accumulation in the plant as well as the fatty acid (FA) profiles of the plant cell membrane. Under the same growing conditions, the nCuO exposure resulted in more Cu accumulation than did the Cu2+ exposure. Multiple microscopic techniques confirmed the internalization and sequestration of nCuO in root cell vacuoles, where transformation of Cu­(II) to Cu­(I)Cl occurred. Short and long exposures (96 h versus 28 days) to both nCuO and Cu2+ elevated FA saturation degrees in plant cells through oxidative stress, as verified by in situ detection of superoxide radicals, with conversions mostly from C18:3, C16:3, and C18:2 to C16:0. Only the long exposure to nCuO significantly brought about an additional elevation of FA saturation degree in root cells. These results demonstrated that the acute effects of plant exposure to nCuO were mainly produced from the stress of Cu2+ ions released from nCuO dissolution, while the chronic effects in roots were significantly developed by the nCuO particle stress. The findings in this work are novel and may offer significant implications in better understanding nanoparticle-induced phytotoxicity and potential risks in ecosystems.