Down-regulation of leaf N and Rubisco under elevated CO₂ (eCO₂) are accompanied by increased non-structural carbohydrates (NSC) due to the sink–source imbalance. Here, to investigate whether the canopy position affects the down-regulation of Rubisco, we measured leaf N, NSC and N allocation in two species with different heights at maturity [Fraxinus rhynchophylla (6.8 ± 0.3 m) and Sorbus alnifolia (3.6 ± 0.2 m)] from 2017 to 2019. Since 2009, both species were grown at three different CO₂ concentrations in open-top chambers: ambient CO₂ (400 ppm; aCO₂); ambient CO₂ × 1.4 (560 ppm; eCO₂1.4); and ambient CO₂ × 1.8 (720 ppm; eCO₂1.8). Leaf N per unit mass (N_mass) decreased under eCO₂, except under eCO₂1.8 in S. alnifolia and coincided with increased NSC. NSC increased under eCO₂ in F. rhynchophylla, but the increment of NSC was greater in the upper canopy of S. alnifolia. Conversely, Rubisco content per unit area was reduced under eCO₂ in S. alnifolia and there was no interaction between CO₂ and canopy position. In contrast, the reduction of Rubisco content per unit area was greater in the upper canopy of F. rhynchophylla, with a significant interaction between CO₂ and canopy position. Rubisco was negatively correlated with NSC only in the upper canopy of F. rhynchophylla, and at the same NSC, Rubisco was lower under eCO₂ than under aCO₂. Contrary to Rubisco, chlorophyll increased under eCO₂ in both species, although there was no interaction between CO₂ and canopy position. Finally, photosynthetic N content (Rubisco + chlorophyll + PSII) was reduced and consistent with down-regulation of Rubisco. Therefore, the observed N_mass reduction under eCO₂ was associated with dilution due to NSC accumulation. Moreover, down-regulation of Rubisco under eCO₂ was more sensitive to NSC accumulation in the upper canopy. Our findings emphasize the need for the modification of the canopy level model in the context of climate change.