As the largest contiguous karst area of China, the southwestern karst area is a typical ecologically fragile area affecting local vegetation dynamics. Ecosystem water use efficiency (WUE) is an important factor outlining the vegetation’s ability to produce organic matter with a limited water supply. Therefore, it is important to determine WUE variation trends in this ecologically fragile region. In this paper, we used MODIS remote sensing datasets, meteorological data and land cover data to analyze the spatiotemporal changes in vegetation water use efficiency in the southwest karst region from 2001 to 2017. We also further quantitatively analyzed the impact of climate change and human activities on the spatial and temporal patterns of vegetation WUE in the study area. The main conclusions were as follows. (1) From 2001 to 2017, in terms of temporal characteristics, the interannual variation in WUE fluctuated greatly, ranging from 1.33 to 1.51 g C kg-1 H2O, with a multiyear average of 1.43 g C kg-1 H2O and an average rate of change of -0.0046 g C kg-1 H2O yr-1. In terms of spatial characteristics, areas with a higher WUE were concentrated in central Sichuan and northeastern Yunnan. (2) There were also certain differences in the WUE for different vegetation types. The annual average WUE of each vegetation type decreased in the following order: evergreen coniferous forest> evergreen broad-leaved forest> mixed forest> deciduous broad-leaved forest> cultivated land> deciduous coniferous forest> grassland> cultivated land and natural vegetation> shrub forest. (3) The vegetation WUE of 70.66% in this area was positively correlated with temperature. Additionally, 79.68% of the vegetation WUE was negatively correlated with precipitation. The relative contribution rates of climate change and human activities to the change trend in WUE were 15% and 85%, respectively. These results provide scientific support for local vegetation restoration and protection policies.