There are special climate and terrain in mountain areas of Guizhou Plateau including less sunshine,more cloud,rain and complex underlying surface. It is important for further understanding the process of carbon and water cycle in Karst area if we accurately explore the spatio-temporal differential characteristics of ecosystem water use efficiency (WUE) in this area. The data of meteorological stations in this paper were handled with interpolation by ANUSPLIN software. Meanwhile,high-quality MODIS-NDVI data had been reconstructed by time series harmonic analysis and quality control file as common input data of the CASA model to simulate NPP and estimated WUE with MOD16-ET data. With statistical method including Theil-Sen Median trend analysis,Mann-Kendall test and stability analysis,the spatial and temporal differential characteristics of WUE in different landform regions and vegetation types of Guizhou Plateau from 2000 to 2014 were compared and analyzed. The following were the results: (1) The spatial pattern for trend and WUE average value in the researching area during past 15 years was higher in western part than eastern and the trend of WUE was increasing significantly (R2=0.63,P < 0.001). (2) From the point of view of various landform regions,the order of WUE average value for many years was: fault depression basin > karst canyon > peak-cluster depression > non-karst landform > karst plateau > karst trough valley, among which fault depression basin and karst canyon showed a significant increase trend (P < 0.05). (3) Viewing from different vegetation types,WUE value size ranked as mixed forest > broadleaf forest > cultivated land > coniferous forest > grassland > shrub. Coniferous forest and shrub coefficient variation (CV) were higher,but the former one had stronger resistance to climate fluctuations with faster growth rate. The CV value for grassland was lowest and change rate was slowest which was not easily affected by climate fluctuations. Because of the remarkable effect of ecological projects such as returning farmland to forests,the variation rate of WUE for cultivated land was the fastest.