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Graphical abstract, Nitrate uptake by plants is mediated by specific transport proteins in roots (NRTs), which are also dependent on the activity of proton pumps that energize the reaction. Nitrogen (N) metabolism in plants is sensitive to copper (Cu) toxicity conditions. To understand how Cu affects the uptake and assimilation processes, this study assesses the inhibitory effects of elevated Cu levels on the expression of genes related to N absorption, transport and assimilation in roots of Arabidopsis. Plants were grown hydroponically for 45 days, being exposed to a range of Cu concentrations in the last 72 h or alternatively exposed to 5.0 μM Cu for the last 15 days. High Cu levels decreased the uptake and accumulation of N in plants. It down-regulated the expression of genes encoding nitrate reductase (NR1), low-affinity nitrate transporters (NRT1 family) and bZIP transcription factors (TGA1 and TGA4) that regulate the expression of nitrate transporters. Cu toxicity also specifically down-regulated the plasma membrane proton pump, AHA2, whilst having little effect on AHA1 and AHA5. In contrast, there was an up-regulation of high-affinity nitrate transporters from the NRT2 family when exposed to medium level of Cu excess, but this was insufficient for restoring N absorption by roots to control levels. These results demonstrate that plants display specific responses to Cu toxicity, modulating the expression of particular genes related to nitrate uptake, such as low-affinity nitrate transporters and proton pumps.