The atmospheric CO2 concentration ([CO2]) is increasing and predicted to reach ∼550ppm by 2050. Increasing [CO2] typically stimulates crop growth and yield, but decreases concentrations of nutrients, such as nitrogen ([N]), and therefore protein, in plant tissues and grains. Such changes in grain composition are expected to have negative implications for the nutritional and economic value of grains. This study addresses two mechanisms potentially accountable for the phenomenon of elevated [CO2]-induced decreases in [N]: N uptake per unit length of roots as well as inhibition of the assimilation of nitrate (NO3-) into protein are investigated and related to grain protein. We analysed two wheat cultivars from a similar genetic background but contrasting in agronomic features (Triticum aestivum L. cv. Scout and Yitpi). Plants were field-grown within the Australian Grains Free Air CO2 Enrichment (AGFACE) facility under two atmospheric [CO2] (ambient, ∼400ppm, and elevated, ∼550ppm) and two water treatments (rain-fed and well-watered). Aboveground dry weight (ADW) and root length (RL, captured by a mini-rhizotron root growth monitoring system), as well as [N] and NO3- concentrations ([NO3-]) were monitored throughout the growing season and related to grain protein at harvest. RL generally increased under e[CO2] and varied between water supply and cultivars. The ratio of total aboveground N (TN) taken up per RL was affected by CO2 treatment only later in the season and there was no significant correlation between TN/RL and grain protein concentration across cultivars and [CO2] treatments. In contrast, a greater percentage of N remained as unassimilated [NO3-] in the tissue of e[CO2] grown crops (expressed as the ratio of NO3- to total N) and this was significantly correlated with decreased grain protein. These findings suggest that e[CO2] directly affects the nitrate assimilation capacity of wheat with direct negative implications for grain quality.