The stimulation effect of elevated [CO₂] (e[CO₂]) on plant growth is modulated by nitrogen (N) availability, yet the mechanisms of this modulation under patchy N supply remain largely elusive. This study aimed to investigate the mechanisms by which patchy nutrient supply in the root-zone influence on N uptake, partitioning and use efficiency of tobacco plants (Nicotiana tabacum L.) grown under e[CO₂]. A split-root pot experiment was conducted on tobacco plants grown at ambient (a[CO₂], 400 μmol L⁻¹) and e[CO₂] (800 μmol L⁻¹) conditions during anthesis. Plants were subjected to two fertilizer levels [0 and 113-29-214 (N-P-K) mg kg⁻¹ soil] and three fertilization regimes (partial root-zone fertilization, PRN, couple root-zone fertilization, CRN and no fertilization, CK). Elevated [CO₂] significantly decreased N concentrations in all tobacco organs, with the greatest reduction in leaves. Positive responses of tobacco biomass and NUE to e[CO₂] were recorded, and a large amount of ¹⁵N labelled fertilizer-N was partitioned to stems at the expense to leaves. Compared to the partially fertilized root, an equal N concentration was observed in the non-fertilized root. In addition, compared with CRN, PRN increased the root exudates of sugar and organic acid; however, the increased root biomass by nutrient patchiness did not enhance plant total N uptake. Collectively, e[CO₂] could sustain N assimilation and distribution of tobacco plants in response to natural heterogeneous nutrient available in the soil caused by patchy fertilization.