In addition to soil characteristics, two plant traits control the supply of water from the soil to the canopy: root growth and plant hydraulic conductance. Here we examine the impact of root growth and hydraulic conductance on water uptake and transpiration of walnut under deficit irrigation. A greenhouse experiment was conducted using nine young walnut trees (Juglans regia L.) grown for three months in transparent pots, equipped with: (i) rhizotron tubes, which allowed for non-invasive monitoring of root growth; (ii) pressure transducer tensiometers, recording soil water potential at soil-root interfaces; (iii) psychrometers attached to mature leaves, measuring stem water potential; and (iv) weighing scales used to determine total plant transpiration. Irrigation treatments consisted of different replenishment levels (100%, 75%, and 50%) of potential transpiration replicated over time. Walnut trees showed rapid physiological acclimation characterized by a fast decline and subsequent stabilization of transpiration rates soon after the beginning of drought stress treatments. We also observed a significant decrease in plant hydraulic conductance with decreasing soil and stem water potential under drought stress. At the end of the experiment, isotopic measurements revealed the integrated effect of physiological acclimation on canopy carbon-water relations. Leaf carbon isotope ratios showed significant increases in water-use efficiency with deficit irrigation levels. Leaf water hydrogen and oxygen isotope ratios confirmed that changes in water use-efficiency were caused by decreases in transpiration. Conversely, root growth was highest under low stress (T100) and lowest under high stress (T50). These results indicate the existence of a fundamental tradeoff between water-use efficiency and root growth, which will be useful to optimize the application of water and improve the design of irrigation systems in walnut orchards.