The lower Jinsha River Basin (LJRB) is an important protected area in the regional environments and ecosystems in the southwest of China. It is also a key region for future hydroelectric development and is of great strategic significance for national economy and regional sustainable development. Although the LJRB is one of vital energy bases in China for its huge potential hydropower resources, the region's ecosystems are very fragile. In order to protect these ecologically fragile environments, eco-regionalization based on ecological function at national scale has been recently formulated, promulgated, and implemented; however, only few studies have addressed ecological regionalization at basin scale. It was assumed that the eco-regionalization at the national scale could not be applied equally and accurately to the local eco-regionalization. Therefore, we tested weather national eco-regionalization could be used at the small regional scale, and sought appropriate eco-regionalization plan applicable at a specific scale. This research on regionalization based on ecological function was carried out in the LJRB by incorporating spatial analysis methods in remote sensing and GIS and qualitative and quantitative analytic hierarchy process analyses in the process of regionalization in accordance with the local climate, topography, and ecological environment. The LJRB was divided into two eco-regions and six sub-eco-regions composed of 19 eco-functional zones. Furthermore,19 critical eco-functional zones were delineated by synthetically analyzing dominant eco-functions, involving conservation of water and soil, and conservation of biodiversity in the LJRB. The results indicated that regionalization in the LJRB is very important for the local ecological sustainability. This study will help to exploit and allocate natural resources orderly, rationally guide the land planning for industries, as well as provide scientific support for protecting the environment and maintaining ecological security of hydroelectric development in the LJRB.