Water use efficiency (WUE) of an ecosystem is an important index to measure the degree of coupling of the carbon and water cycles. The WUE of a cropland ecosystem in Anhui Province was estimated and the spatio-temporal distribution of cropland WUE was analyzed to explore its influencing factors, which will be of great significance for scientific allocation and rational utilization of water resources in the cropland region. This study focused on the application of data from moderate-resolution imaging spectroradiometer (MODIS) and meteorological data, estimated the cropland WUE, analyzed the spatio-temporal characteristics of cropland WUE in Anhui Province over the last 15 years, and evaluated the relationship of WUE with climatic factors. The results showed that 1) Between 2000 and 2014, the average annual cropland WUE for Anhui Province was 1.54 gC mm^-1 m^-2, ranging from 1.38 to 1.66 gC mm^-1 m^-2. As a whole, the annual growth trend of cropland WUE was 0.011 gC mm^-1 m^-2, and cropland WUE had a strong spatial variability in Anhui Province, where it was higher in the north and lower in the southern part of the province. The cropland WUE north of the Huaihe River was higher than that of the province as a whole, while that of the Huaihe River was lower than the province average. 2) The variation of cropland WUE in Anhui Province showed a bimodal change pattern with obvious seasonal variation, in the order of spring > autumn > summer > winter. Taking Huaihe as the boundary, the changing trends of the cities in the north and the south were slightly different. The maximum value south of Huaihe was in April, while that north of Huaihe was the highest in March. 3) The dynamic change of cropland WUE in Anhui Province was closely related to temperature and rainfall. The regions with cropland WUE changes resulting from non-climate factors made up 81.42% of the total area of cropland in the province, followed by that mainly affected by precipitation (17.14%) and temperature (0.73%), while the combination of precipitation and temperature had the smallest impact on water use efficiency (0.71%). Overall, rainfall played the most important role of all climatic factors in the changes in cropland WUE, but due to the influence of human factors, non-climate factors had a greater impact on cropland WUE changes.