Water availability is a key ecological factor for plant growth in arid and semi-arid regions. We identified the characteristics of water-uptake patterns by Achnatherum splendens and how it responds to changes in water conditions in typical steppe ecosystem (Lake Qinghai watershed). We analyzed stable isotopes of plant stem water and potential water sources including soil water, groundwater, and precipitation via direct comparison with differences among potential water sources and a multi-source mixing model calculating the plant-use proportions from diverse soil depths under natural and drought treatment areas. The results showed that there were large fluctuations in the soil moisture and the isotopic contents (δD) of soil water in the surface soil layers, directly influenced by precipitation and evaporation. The slope and intercept of the evaporative soil water line were evidently smaller than those of the meteoric water line, indicating that the isotopic contents from soil water experienced stronger evaporation fractionation processes. The isotopic contents (δD and δ¹⁸O) of the stem were scattered along the soil evaporative water line indicating that the possible water sources were extracted from diverse soil water depths. A. splendens absorbed mainly surface soil water (the proportion from 0-10cm was 45.1% in June) at the early growth stage. Large precipitation events significantly affected the soil water content and isotopic contents and infiltrated into deep soil layers. Differences in plant water use proportions were minimal during August and September, which revealed that the roots of A. splendens extracted water from various depths when soil water availability was high. Under the drought treatment conditions, A. splendens used mostly surface soil water (0-30 cm) in early July. The absorption water depth shifted to deep soil layers when the surface soil water became drier. However, the absorption depth again shifted from deep to surface soil layers when surface soil moisture sharply increased after irrigation. Groundwater was not a main water source for A. splendens during the entire growing season.