Three-River Headwaters region is a critical water conservation area in China and an ecological barrier of Southeast Asia, which has an important ecological strategic position. It is important to reveal the spatial distribution, changing trends and influencing factors of water conservation capacity to promote ecological protection. It is also meaningful in improving the regional water supply capacity and maintaining the stability of the ecosystem. Based on the InVEST model, this paper estimated the spatial and temporal changes of water conservation capacity and analyzed the influencing factors. The results indicated that the grassland ecosystem accounted for most of the water conservation, with an average annual amount of water conservation of 12.00 billion m³. From 1990 to 2020, the water conservation in Three-River Headwaters region was significantly rising with an increasing rate of 0.18 billion m³/a (P<0.05). The average amount of water conservation was 16.38 billion m³. From 1990 to 2005, the increasing rate of water conservation was higher than that from 2005 to 2020. The spatial distribution of the water conservation capacity was high in the southeast and low in the northwest. The significant growth area was 220700 km², accounting for 60.79% of the total area of the region. Before the implementation of ecological project (1990-2005), climate changes such as increased precipitation, increased actual evapotranspiration and decreased actual evapotranspiration ratio were the main factors driving the increase in water conservation capacity. After the implementation of ecological project (2005-2020), land use/cover changes such as increased forest and grassland areas were the main factors driving the increase in water conservation capacity.