Mixed forests typically exhibit higher productivity but also greater water consumption, making understanding their water use patterns across developmental stages essential for sustainable management. This study investigated water uptake strategies in mixed plantations of Betula alnoides and Castanopsis hystrix aged seven, 15, and 22 years in southern China. Stable isotopes of hydrogen (2H) and oxygen (18O) in soil and xylem water were analyzed across dry and rainy seasons to identify water sources, while leaf delta 13C measurements assessed water use efficiency (WUE). Root morphology and distribution were also examined. With increasing stand age, both species absorbed more water from deeper soil layers, especially during dry seasons. B. alnoides shifted its main water source from 0-20 cm at age seven to 40-60 cm at ages 15- and 22-, while C. hystrix transitioned to the 20-40 cm layer. During rainy seasons, both species utilized more shallow water, particularly C. hystrix. Roots of both species were mainly distributed in the 0-40 cm soil layer, accounting for 66-85% of B. alnoides roots and 67-70% of C. hystrix roots. Root systems expanded significantly with age, with B. alnoides developing deeper and wider roots and C. hystrix exhibiting higher specific root length and surface area. Both species exhibited higher dryseason WUE in their younger mixed plantations across all ages than in older ones, and B. alnoides generally demonstrated greater WUE than C. hystrix, indicating better adaptation to water variability. The complementary water use strategies and rooting patterns between species supported positive mixing effects in soil water utilization. These findings offer a theoretical basis for guiding mixed-species afforestation and ecological management in warm subtropical regions.