Ecosystem services related to water supply is currently topical in ecology. Water supply service is essential for internal water circulation and water balance in river basins. Dongjiang Lake Basin is a national key basin and pilot project of water resource ecological compensation and the water supply service is a basic ecosystem service in the basin. This paper evaluated the water supply service and analyzed the spatiotemporal patterns and variation characteristics from 1995 to 2010 based on the InVEST model from the scale of basin and subbasins, and compared water supply service capacity of different land cover types. (1) In 1995-2010, the water yield in the basin decreased after an initial increase with the annual water yield between 1000 and 1500 mm and the value in 2000 was the highest. (2) The spatial distribution patterns of different years were similar with water yield increasing from the lake to the surrounding area. (3) The water yield, with an average of 1600 mm, was high in urban and built-up areas, herbaceous steppe, shrub forest, and shrub grassland, while it was less than 800 mm in the area of bottom lands, water bodies, and paddy fields. (4) The water yield in each sub-basin was 750-1700 mm and the water supply capacity increased from the lake to the surrounding area. The subbasins in the northeast were the highest water yield areas of the whole basin. From the perspective of water balance, precipitation and actual evapotranspiration are two key factors determining the water yield. Spatiotemporal variation of the amount of water supply in Dongjiang Lake Basin resulted from interactions between climate and land cover change. The present study provides guidelines for the efficient management of water resources in the Dongjiang Lake Basin. It is of vital practical significance to improve the ecosystem service function and ecological environmental quality, and to promote the sustainable development of the social economy.