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Rapid and periodic assessment of the impact of land cover change and climate variability on ecosystem services at regional levels is essential to understanding services and sustainability of ecosystems. This study focused on quantifying and assessing the changes in multiple ecosystem services in the Three-River Headwaters Region (TRHR), China in 2000â2012. Based on the widely used biophysical models including Integrated Valuation of Ecosystem Services and Trade-Offs (InVEST), Revised Wind Erosion Equation (RWSQ), and Carnegie-Ames-Stanford Approach (CASA) models, this study assessed the historical flow of regulating services, including soil conservation, water yield, and carbon sequestration, and provisioning service food provision. The soil conservation function of ecosystem was slightly enhanced as a whole, and water yield increased sharply, with both the soil conservation and water yield showing an increasing spatial homogenization. The net primary productivity (NPP) and food production increased substantially from 2000 to 2012. Ecosystem services are closely and complexly interlinked. The correlation analyses indicated a trade-off between the water yield and carbon sequestration, however, a synergy between soil conservation and carbon sequestration. Congruence between the three different ecosystem provisioning services, including pasture, meat, and grain, was found. There was also a synergy between food production and ecosystem carbon sequestration in the TRHR. Climatic variability and vegetation restoration are important for the ecosystem services flow. Correlation analyses showed that the increase in precipitation significantly enhanced the water yield (P