Water supply is an important freshwater ecosystem service provided by ecosystems. Water shortages resulting from spatio-temporal heterogeneity of climate condition or human activities present serious problems in the Guizhou Province of southwest China. This study aimed to analyze the spatio-temporal changes of water supply service using the Integrated Valuation of Ecosystem Services and Tradeoffs (InVEST) model, explore how climate and land-use changes impact water supply provision, and discuss the impact of parameters associated with climate and land-use in the InVEST model on water supply in the region. We used data and the model to forecast trends for the year 2030 and found that water supply has been declining in the region at the watershed scale since 1990. Climate and land-use change played important roles in affecting the water supply. Water supply was overwhelmingly driven by the reference evapotranspiration and annual average precipitation, while the plant evapotranspiration coefficients for each land-use type had a relatively small effect. The method for sensitivity analysis developed in this study allowed exploration of the relative importance of parameters in the InVEST water yield model. The Grain-for-Green project, afforestation, and urban expansion control should be accelerated in this region to protect the water supply.