Water availability is one of the most important factors affecting vegetation distribution in terrestrial ecosystems, especially in arid regions. Plant species vary in their abilities to absorb water from different soil depths.The depth from which plant species obtain water directly determines their distributions. Therefore, quantifying the depth of water uptake of dominant species is critically important to defining and predicting vegetation spatiotemporal distributions under global change. Stable isotopes of oxygen and hydrogen have become an important tool for identifying the plant water uptake sources; this is because various water sources possess distinct oxygen or hydrogen isotope signatures. However, there has been little research on shifts in water sources with phenological development. Research on the mean depth of water uptake of trees of different ages is essential for formulating conservation strategies for the riparian tree, Populus euphratica. This study assessed the contributions of different potential water sources to P. euphratica. We used the Romero-Saltos model and levels of stable oxygen and hydrogen isotopes (δD, δ¹⁸O) in the xylem of P. euphratica individuals of different ages and in soil water and groundwater along the lower Heihe River. For all the three age classifications (young forest, mature forest and over-mature forest), the shallowest soil water sample (5 cm in depth) had the highest or near highest measured values of δ¹⁸O: 4.4 ‰, 4.1 ‰, and 1.5 ‰, respectively. δD and δ¹⁸O decreased with increasing depth. The greatest rates of decrease occurred near the surface, reflecting evaporation of soil water near the surface. The similarity between deep soil and groundwater δD and δ¹⁸O values of for all forest ages strongly suggests that the deep soil water is derived from groundwater. We found significant differences in δ¹⁸O values in the xylem among different ages of P. euphratica. The δ¹⁸O values of young, mature and over-mature forest reached -5.37‰, -6.03‰, and -6.92‰ respectively, reflecting the reliance of older trees on deeper sources of water with lower δ¹⁸O values, closer to those of groundwater. The mean depth of water uptake P. euphratica varied with age. The mean depths of water uptake of seedlings, mature and over-mature forest were 37, 145, and 149.5 cm, respectively. This indicates that P. euphratica accesses deeper soil moisture with age. The δ¹³C values of young, mature and over-mature forest were -26.30‰, -27.53‰, and -29.03‰, respectively. Similarly, we found significant differences in δ¹³C values of leaves among different ages of P. euphratica. The water use efficiency of young, mature, and over-mature forest were different, and tended to decrease with age. For all ages of forest, soil moisture peaked between 100 and 200 cm depth, which it is close to the mean water uptake depth of P. euphratica of all ages.