Abstract: Excessive copper concentrations affect nutrient uptake in sensible species. However, the effects of copper on nutrient uptake in tolerant species have not been studied equally well. Thus, the main objective of this study was to determine the effect of rising Cu exposure on nutrient uptake rates and root/shoot nutrient contents in two species with distinct copper tolerance: Acacia caven (Mol.) Mol. and Helianthus annuus L. To this end, a hydroponic experiment was conducted. Copper treatments were applied at concentrations of 0, 2.0, 3.9, 7.9, and 15.7 μM. Relative root elongation was chosen as the endpoint for Cu toxicity assessment. The results of our study demonstrate that A. caven and H. annuus differ greatly in their tolerance to Cu. Copper concentration in the solution associated with the calculated EC25 was found to be six times higher in A. caven (15 μM) than in H. annuus (2.3 μM). When effective concentrations were instead based on the measured root Cu concentrations, the difference observed was ten times greater (1044 and 98 mg/kg for A. caven and H. annuus, respectively). Both species showed equal Cu uptake kinetics, with root absorbing power (α) of 7.5 ± 0.7 × 10–6 and 7.8 ± 0.5 × 10–6 cm s–1 for A. caven and H. annuus, respectively. Rising Cu concentrations in the exposure solution progressively diminished the influx of Ca into the roots of H. annuus, whereas no significant effect was noted for A. caven.