Hydrological niche segregation (HNS) is widely recognized as a key factor in species coexistence, but experimental evidence on water resource allocation among coexisting shrubs in desert ecosystems remains limited. We investigated the dynamics of hydrological niche segregation between two dominant desert shrubs (Nitraria sphaerocarpa and Reaumuria songarica) by analyzing stable isotope compositions (delta 2H, delta 18O) and soil water availability. The results showed that coexisting species exhibit distinct and flexible water use strategies. N. sphaerocarpa primarily utilizes shallow soil moisture during average precipitation periods but shifts to deeper water sources under arid conditions, with its distribution range spatially overlapping with that of R. songarica. Both coexisting species responded only to larger precipitation pulses, but N. sphaerocarpa exhibited higher uptake intensity and longer reliance duration on shallow water sources post-precipitation. As interannual precipitation decreases, the niche overlap between coexisting species significantly increases, while the niche width of R. songarica expands by 1.11 % and that of N. sphaerocarpa contracts by 3.24 %. These results highlight HNS as a dynamic mechanism that promotes coexistence through flexible resource partitioning. Under climate change scenarios, the plasticity of HNS is crucial for maintaining biodiversity in arid regions.