【Objective】Understanding the water use patterns and dynamic changes of sand-fixing plants is crucial for scientifically guiding the sustainable development of artificial vegetation and the efficient use of water resources in desert ecosystems in arid and semi-arid regions. However, in water-limited desert ecosystems, there is still a lack of in-depth understanding of the seasonal variation in water sources of different sand-fixing plants. This study was designed to examine the root water source and hydrological niche segregation of artificially restored plants in the Mu Us Sandy Land, aiming to provide scientific bases for the rational configuration of artificial vegetation and sustainable use of water resources in desert ecosystems.【Method】This study focused on three typical sand-fixing plants in the Gechougou catchment in the northeastern margin of the Mu Us Sandy Land, northwest China, including Pinus sylvestris var. mongolica (P. sylvestris), Salix psammophila (S. psammophila), and Amygdalus pcdunculata Pall (A. pedunculata). The plant xylem water and their potential water sources (rainwater and soil water at different depths) were collected monthly during three consecutive growing seasons (May—October) of 2019—2021. δ²H and δ¹⁸O of xylem water were measured by Isotope Ratio Mass Spectrometry (IRMS; MAT253, Thermo Fisher Scientific, Germany) to mitigate interference from trace organic compounds. δ²H and δ¹⁸O of rainwater and extracted soil water were measured by Laser-Based Liquid Water Isotope Analyzer (LWIA; Picarro L2130i, USA). The vertical distribution of fine root (<2 mm) biomass and soil water content in each species stand were also determined. The MixSIAR model was used to quantitatively analyze the seasonal changes in plant water sources, and the proportional similarity index (PSI) was employed to quantify the degree of hydrological niche separation and overlap. 【Result】 Results indicated that the water use patterns of P. sylvestris, S. psammophila, and A. pedunculata exhibited significant seasonal variations. During the dry season, P. sylvestris and S. psammophila primarily absorbed water from the 120～300 cm soil layer (63.43±8.42% and 56.12±1.23%, respectively), while A. pedunculata mainly obtained water from 40～200 cm soil layer (54.00±4.16%). In the rainy season, all three species mainly absorbed water from the 0～120 cm soil layer (57.18±5.84%, 60.78±15.00%, and 60.43±6.80%, respectively). Compared with the dry season, P. sylvestris, S. psammophila, and A. pedunculata significantly increased their absorption of water from the 0–40 cm soil layer during the rainy season (P<0.05). The changes in water use patterns of different sand-fixing plants were closely related to their root vertical distribution and the seasonal variation of soil water availability. P. sylvestris and A. pedunculata exhibited relatively low hydrological niche overlap (PSI<0.75), suggesting minimal competition for the same water sources, making them suitable for interplanting. In contrast, P. sylvestris and S. psammophila showed very similar water use strategies throughout the growing season, displaying significant water competition. 【Conclusion】The findings of this study highlight the importance of considering species-specific water use patterns when planning vegetation restoration efforts. Specifically, our results support the idea of strategically interplanting species with complementary hydrological niches to enhance vegetation resilience, improve water use efficiency, and promote sustainable ecosystem restoration in arid and semi-arid desert regions.