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 (eCO2). This study investigated the effect of drought in shallow soil versus sub-soil on agronomic and physiological responses of wheat to eCO2 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 (aCO2, ∼400 μmol mol-1) or eCO2 (∼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 aCO2, net assimilation rate was higher and stomatal conductance was lower under eCO2, 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 eCO2-stimulation of root growth especially in the well-watered bottom layer. We suggest that stimulation of belowground biomass under eCO2 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, eCO2 may help mitigating the effect of drying surface soil.