Water is the main limiting factor of vegetation growth in arid and semi-arid area, and water use is a key ecohydrological process reflecting the response of vegetation to the environmental changes. At present, there is a lack of systematic analysis of the differences in water use characteristics of plants in different families and genera of grasslands in arid and semi-arid area, and the quantitative relationship between plant water use characteristics and environmental factors needs to be further studied. In this study, a transect survey was conducted in the desert steppe of the Inner Mongolia. Soil sample at a depth of 0-100 cm, junctions (stems) and leaf samples were collected from 7 plots, and the isotopes of δ²H and δ¹⁸O in soil water and plant water and plant leaf δ¹³C were determined. The proportion of water sources of different families and genera plants were determined by the MixSIAR model, and the differences of water use sources and water use efficiency of plants of different families and genera were analyzed, and the quantitative relationship between the proportion of plant water sources and water use efficiency and various environmental factors were established. The results showed that Gramineae and Compositae mainly used soil water at a depth of 0-30 cm, which the absorption ratio were respectively 55.63% and 51.84%. Their leaf δ¹³C that were -26.61‰ and -27.91‰ were lower than those of other families (including Tamaricaceae, Chenopodiaceae, and Rosaceae), which the leaf δ¹³C was -26.36‰. The absorption ratio of other families that mainly used soil water at a depth of 60-100 cm was 36.83% and the water use strategies were conducive to survival in drought conditions. The proportion of soil water utilization at a depth of 0-30 cm by Stipa was 55.28%, and the leaf δ¹³C was -26.38‰, which were higher than that of Artemisia Linn (31% and -28.14‰). The water use strategy of Stipa made it have strongly ecological adaptability in arid environment. The response of soil water utilization ratio at different depths and leaf δ¹³C to different environmental factors were different, among which the proportions of plant water sources were mainly affected by the average annual rainfall, average annual temperature, and leaf circumference (P<0.05). This study revealed the water use characteristics of plants in desert grasslands, which was of great significance for understanding the response strategies of plants to environmental change, and it can provide the scientific basis for ecosystem management in this region.