Engineered nanoparticles (ENPs) exhibit unique properties advantageous in a number of applications, but they also represent potential health and environmental risks. In this study, we investigated the phytotoxic mechanism of CuO ENPs using transcriptomic analysis and compared this response with the response to CuO bulk particles and ionic Cu2+. Ionic Cu2+ at the concentration of 0.16 mg L-1 changed transcription of 2692 genes (p value of <0.001, fold change of ≥2) after 7 days of exposure, whereas CuO ENPs and bulk particles (both in the concentration of 10 mg L-1) altered the expression of 922 and 482 genes in Arabidopsis thaliana roots, respectively. The similarity between transcription profiles of plants exposed to ENPs and ionic Cu2+ indicated that the main factor in phytotoxicity was the release of Cu2+ ions from CuO ENPs after 7 days of exposure. The effect of Cu2+ ions was evident in all treatments, as indicated by the down-regulation of genes involved in metal homeostasis and transport and the up-regulation of oxidative stress response genes. ENPs were more soluble than bulk particles, resulting in the up-regulation of metallochaperone-like genes or the down-regulation of aquaporins and metal transmembrane transporters that was also characteristic for ionic Cu2+ exposure.