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Lakes are important for global ecological balance and provide rich biological and social resources. However, lake systems are sensitive to climate change and anthropogenic activities. Poyang Lake is an important wetland in the middle reach of the Yangtze River, China and has a complicated interaction with the Yangtze River. In recent years, the water level of Poyang Lake was altered dramatically, in particular showing a significant downward trend after the operation of the Three Gorges Dam (TGD) in 2003, thus seriously affecting the lake wetland ecosystem. The operation of the TGD changed both the hydrological regime and the deeper channel in the middle reach of the Yangtze River, and affected the river-lake system between the Yangtze River and Poyang Lake. This study analyzed the change in the water level of Poyang Lake and quantified the contributions of the TGD operation, from the perspectives of water storage and erosion of the deeper channel in the middle reach of the Yangtze River, through hydrodynamic model simulation. The erosion of the deeper channel indicated a significant decrease in annual water level. However, due to the water storage of the TGD in September and October, the discharge in the Yangtze River sharply decreased and the water level of Poyang Lake was largely affected. Especially in late September, early October, and mid-October, the contributions of water storage of the TGD to the decline in the water level of Poyang Lake respectively reached 68.85%, 59.04%, and 54.88%, indicating that the water storage of the TGD was the main factor in the decrease in water level. The erosion of the deeper channel accelerated the decline of the water level of Poyang Lake and led to about 10% to 20% of the decline of water level in September and October. Due to the combined operation of the TGD and more reservoirs under construction in the upper TGD, the long-term and irreversible influence of the TGD on Poyang Lake should be further explored in the future.