Through stimulation of root growth, increasing atmospheric CO2 concentration ([CO2]) may facilitate access of crops to sub-soil water, which could potentially prolong physiological activity in dryland environments, particularly because crops are more water use efficient under elevated [CO2] (e[CO2]). This study investigated the effect of drought in shallow soil versus sub-soil on agronomic and physiological responses of wheat to e[CO2] in a glasshouse experiment. Wheat (Triticum aestivum L. cv. Yitpi) was grown in split-columns with the top (0–30 cm) and bottom (31–60 cm; ‘sub-soil’) soil layer hydraulically separated by a wax-coated, root-penetrable layer under ambient [CO2] (a[CO2], ∼400 μmol mol-1) or e[CO2] (∼700 μmol mol-1) [CO2]. Drought was imposed from stem-elongation in either the top or bottom soil layer or both by withholding 33% of the irrigation, resulting in four water treatments (WW, WD, DW, DD; D = drought, W = well-watered, letters denote water treatment in top and bottom soil layer, respectively). Leaf gas exchange was measured weekly from stem-elongation until anthesis. Above-and belowground biomass, grain yield and yield components were evaluated at three developmental stages (stem-elongation, anthesis and maturity). Compared with a[CO2], net assimilation rate was higher and stomatal conductance was lower under e[CO2], resulting in greater intrinsic water use efficiency. Elevated [CO2] stimulated both above- and belowground biomass as well as grain yield, however, this stimulation was greater under well-watered (WW) than drought (DD) throughout the whole soil profile. Imposition of drought in either or both soil layers decreased aboveground biomass and grain yield under both [CO2] compared to the well-watered treatment. However, the greatest ‘CO2 fertilisation effect’ was observed when drought was imposed in the top soil layer only (DW), and this was associated with e[CO2]-stimulation of root growth especially in the well-watered bottom layer. We suggest that stimulation of belowground biomass under e[CO2] will allow better access to sub-soil water during grain filling period, when additional water is converted into additional yield with high efficiency in Mediterranean-type dryland agro-ecosystems. If sufficient water is available in the sub-soil, e[CO2] may help mitigating the effect of drying surface soil.