The shortage of water resources is a key factor limiting the sustainability of the economy and society. Most of the 25 National Key Ecological Function Areas (NKEFAs) in China serve as a source and supplementation for numerous rivers and playing an important role in water resource conservation. Based on the analysis of eco-environmental quality changes in NKEFAs, this study analyzed the spatial pattern of water conservation services in 2000 and 2010 by using a water balance equation. The results indicate that the land cover type of NKEFAs was dominated by grassland, and the proportion of ecological land conversion to non-ecological land (0.3%) was higher than that of non-ecological land conversion to ecological land (0.21%). The fractional vegetation coverage (FVC) and biomass density of NKEFAs gradually decreased from southeast to northwest. The FVC of the Changbai Mountain Forest Function Area (CBS) was the highest, while the biomass density and total biomass were highest in mountain areas in the Middle of Hai'nan Island (HND) and in the Great Khingan and Lesser Khingan Mountains (XAL) respectively. The FVC and biomass of NKEFAs mostly increased in 2000-2010. Water conservation amounts of NKEFAs decreased from southeast to northwest. The average water conservation and total water conservation amount of Nanling Mountain (NL), Guangxi-Guizhou-Yunnan (GQD), and the Wuling Mountain Function Area (WLS) were the highest, while the Yinshan Mountain (YS), Alkin Grassland (AEJ), and the Qilian Mountain Function Area (QLS) had the lowest values. In 2000-2010, the water conservation service of 60% of NKEFAs decreased. Spatial and temporal differences in water conservation services are the result of a combination of ecological environment quality and meteorological conditions. Protection of the ecological environment and vegetation coverage improvement should be strengthened to enhance the function of water conservation. © 2018 by the authors.