Elevated [CO2] (eCO2) can lead to photosynthetic acclimation and this is often intensified by low nitrogen (N). Despite intensive studies of plant responses to eCO2, the regulation mechanism of primary metabolism at the whole-plant level in interaction with NO3‐ supply remains unclear. We examined the metabolic and transcriptional responses triggered by eCO2 in association with physiological–biochemical traits in flag leaves and roots of durum wheat grown hydroponically in ambient and elevated [CO2] with low (LN) and high (HN) NO3‐ supply. Multivariate analysis revealed a strong interaction between eCO2 and NO3‐ supply. Photosynthetic acclimation induced by eCO2 in LN plants was accompanied by an increase in biomass and carbohydrates, and decreases of leaf organic N per unit area, organic acids, inorganic ions, Calvin–Benson cycle intermediates, Rubisco, nitrate reductase activity, amino acids and transcripts for N metabolism, particularly in leaves, whereas NO3‐ uptake was unaffected. In HN plants, eCO2 did not decrease photosynthetic capacity or leaf organic N per unit area, but induced transcripts for N metabolism, especially in roots. In conclusion, the photosynthetic acclimation in LN plants was associated with an inhibition of leaf NO3‐ assimilation, whereas up-regulation of N metabolism in roots could have mitigated the acclimatory effect of eCO2 in HN plants.