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Root water uptake plays an important role in water transport and carbon cycle among Groundwater–Soil–Plant–Atmosphere–Continuum. The acclimation of crops under elevated carbon dioxide concentrations (eCO₂) depends greatly on their capability to exploit soil water resources. Quantifying root water uptake and its relationship with crop growth under eCO₂ remains challenging. This study observed maize growth subjected to current CO₂ (400 ppm) and eCO₂ (700 ppm) treatments via a device combined with a climate chamber and weighing lysimeters. Root water uptake patterns were determined based on the isotopic tracing technique. The main water uptake depth shifted from 0−20 cm under current treatment to 20−40 cm under eCO₂ at the seedling growth stage. Maize took up 22.7% and 15.4% more soil water from a main uptake depth of 40−80 cm at jointing and tasseling stages in response to eCO₂, respectively. More soil water (8.0%) was absorbed from the 80−140 cm layer at the filling stage under eCO₂. Soil water contributions at the main uptake depth during seedling stage were negatively associated with leaf transpiration rate (<i>T_r</i>), net photosynthetic rate (<i>P_n</i>), and leaf area index (<i>LAI</i>) under both treatments, whereas significant positive correlations in the 40−80 cm layer under current treatment shifted to the 80−140 cm layer by eCO₂. Deep soil water benefited to improve <i>T_r</i>, <i>P_n</i> and <i>LAI</i> under both treatments. No significant correlation between soil water contributions in each layer and leaf water use efficiency was induced by eCO₂. This study enhanced our knowledge of crop water use acclimation to future eCO₂ and provides insights into agricultural water management.