As a vital indicator for measuring the coupled carbon–water cycle of an ecosystem, water use efficiency (WUE) can also reflect the adaptive capacity of plants in different ecosystems. Located in Southwest China, the Li River Basin has a representative karst landform, and the uneven rainfall in the region leads to severe water shortage. In this study, we analyzed the spatial–temporal transformation characteristics of the WUE of the basin and its relationship with different influencing factors from 2001 to 2020 based on a correlation analysis and trend analysis. The main conclusions are as follows: (1) The average value of WUE in the Li River Basin was 1.8251 gC· mm−1·m−2, and it kept decreasing at a rate of 0.0072 gC· mm−1·m−2·a−1 in the past 20 years. With respect to the spatial distribution of the multi-year average of WUE, it exhibits a gradual increasing trend from west to east. (2) Between gross primary productivity (GPP) and evapotranspiration (ET), it was found that ET was the primary influencing factor of WUE. Precipitation was positively correlated with WUE in the Li River Basin, accounting for 67.22% of the total area of the basin. The air temperature was negatively correlated with WUE, and the area was negatively correlated with WUE, accounting for 92.67% of the basin area. (3) The normalized difference vegetation index (NDVI) and leaf area index (LAI) were negatively correlated with WUE, and the proportions of negatively correlated areas to the total area of the basin were similar; both were between 60 and 70%. The growth of vegetation inhibited the increase in WUE in the basin to a certain extent. Regarding Vapor Pressure Deficit (VPD), the proportions of positive and negative correlation areas with WUE were similar, accounting for 49.58% and 50.42%, respectively. (4) The occurrence of drought events and the enhancement in its degree led to a continuous increase in WUE in the basin; for different land cover types, the correlation of the standardized precipitation evapotranspiration index (SPEI) was in the following order from strongest to weakest: grassland > cropland > forest > shrubland.
