There is increasing evidence of environmental change impacts on ecosystem processes and services, yet poor understanding of the relative contributions of land use and climate change to ecosystem services variations. Based on detailed meteorological, hydrological records and satellite data over the Yellow River Source Area (YRSA) in Tibetan Plateau from 1980s to 2008, together with a water-yield module of Integrated Valuation of Ecosystem Services and Tradeoffs (InVEST) model and also a Residual Trends (RESTREND) method, we assessed the water supply variations in YRSA during the past three decades and disentangled the relative contributions of land use and climate change. Results show that water supply significantly decreased from 1980 to 2005 and then increased from 2005 to 2008. The quantity slightly decreased from 283.01 mm in 1980 to 276.95 mm in 1995, 270.12 mm in 2000 and 267.97 mm in 2005, and it then rebounded slightly to 275.26 mm in 2008. The water supply variation ranged from 283.01 mm to 267.97 mm. Climate change contributed dominantly to water supply decrease from 1980 to 1995, which accounts for approximately 64% of the decrease. During 1995 to 2000, land use contributed more and about 58% to the water supply decrease as the intense human activities. From 2000 to 2005, climate change became a positive contribution to the water supply as the increased precipitation, but the land use still contributed negatively. From 2005 to 2008, both climate and land use have positive impacts, but land use contributed about 61% to the water supply increase. The implementation of the Three Rivers Source Area Ecological Protection Project has greatly improved the vegetation coverage conditions and the water retention ability during this period. We recommend that the implementation of ecological projects, grazing policies and artificial improvement of degraded grassland would help to conserve the water retention ability and increase water supply.