Based on the comprehensive analyses of 18 core profiles' sedimentary sequences and lithological characteristics in Jianghan-Dongting Basin of the middle reaches of Yangtze River and the spatial-temporal distribution of archeological sites in this area, we reconstructed the Holocene hydro-environmental evolution of the research area, and its relationship with human activities. The comparison reveals that in 11.5-5.5 ka BP, the water level of rivers and lakes in the middle Yangtze River presented a rising trend, concurrently, under the development of Neolithic culture and rice agricultural activities, human occupation extended from piedmont plain to inner basin plain in the research area. The water level fell in 5.5-4.0 ka BP, meanwhile, the number of human settlements of Qujialing-Shijiahe culture rapidly increased, especially in the inner basin plain. The water level rose again around 4.0 ka BP, and floods spread massively in this period, which led to the decline of Shijiahe culture. The main causes of hydro-environmental evolution in the research area are the fluctuation of sea level and the aggradation of fluvio-lacustrine sediments.