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Changes in the level of the Yangtze River caused by anthropogenic water regulation have major effects on the hydrological processes and water cycle in surrounding lakes and rivers. In this study, we obtained isotopic evidence of changes in the water cycle of Yangtze River during the two drought years of 2006 and 2013. Isotopic evidence demonstrated that the δ18O and δD levels in Yangtze River exhibited high spatial heterogeneity from the upper to lower reaches, which were controlled by atmospheric precipitation, tributary/lake water mixing, damming regulation, and water temperature. Both the slope and intercept of Yangtze River evaporative line (δD = 7.88 δ18O + 7.96) were slightly higher than those of local meteoric water line of Yangtze River catchment (δD = 7.41 δ18O + 6.01). Most of the river isotopic values were located below the local meteoric water line, thereby implying that the Yangtze River water experienced a certain degree of evaporative enrichment on isotopic compositions of river water. The high fluctuations in the isotopic composition (e.g., deuterium excess [d-excess]) in the middle to lower reaches during the initial stage of operation for the Three Gorges Dams (2003-2006) were due to heterogeneous isotopic signatures from the upstream water. In contrast to the normal stage (after 2010) characterized by the maximum water level and largest water storage, a relatively small variability in the deuterium excess was found along the middle to lower reaches because of the homogenization of reservoir water with a longer residence time and complete mixing. The effects of water from lakes and tributaries on the isotopic compositions in mainstream water were highlighted because of the high contributions of lakes water (e.g., Dongting Lake and Poyang Lake) efflux to the Yangtze River mainstream, which ranged from 21% to 85% during 2006 and 2013. These findings suggest that the retention and regulation of the Three Gorges Dams has greatly buffered the isotopic variability of the water cycle in the Yangtze catchment, thereby improving our understanding of the complex lake-river interactions along the middle to lower reaches in the future. [ABSTRACT FROM AUTHOR]