Abstract: Under the combined influence of climate change and regulation of Three Gorges projects, the relationships between Poyang Lake and Yangtze River have been remarkably altered in the middle-lower reaches. A series of existing ecological and environmental problems were induced by the changes of water regimes of Poyang Lake, which have been attracted worldwide attentions. Hence, this study initially investigated the variations of stable water isotopes of precipitation, lake and river water from the channel connecting the Lake Poyang and the Yangtze River, in order to identify the isotopic connections between Yangtze River and Poyang lake water and to analyze the influence of the regulation of Three Gorges projects on the water regimes of Poyang Lake. The results showed that the isotopic compositions of rivers and lakes characterized a remarkably seasonal and spatial distributions, which could be used to reveal the relationship between Poyang Lake and Yangtze River. During the flood regulation period of July, higher δ18O and δ2H values were observed in Poyang lake water compared with Yangtze River water. Moreover, lake water mainly discharged into Yangtze River and its proportion accounted to 75%. During the flood storage dispatching period of November and December, the highest isotopic values were showed due to the stronger enrichment of isotopic compositions. Water level and discharge were obviously changed due to the regulation of Three Gorges projects and affected the relationships between Poyang Lake and Yangtze River. During the flood regulation period, the high water level of the Yangtze River restrained the discharge of lake water. Hence, the water regimes of the Poyang Lake basin should be taken into consideration during the dispatches of the Three Gorges Reservoir, alleviating possible floods in wet seasons and droughts in dry seasons.