Abstract: We explored the temporal variation and attribution characteristics of terrestrial water storage change (T_WSC) in the Yangtze River basin by using Gravity Recovery and Climate Experiment (GRACE) satellite observations and hydrological model simulations and reconstructing and analysing the monthly T_WSC over the Yangtze River basin during 1988—2012. Based on the simulation results of 13 scenarios, we quantitatively separated the relative contributions of climate variability and crucial human activities (including land use changes and reservoir operations) to T_WSC. The main results were as follows: ① The basin-level averaged T_WSC, precipitation, evapotranspiration, and runoff depth showed changing trends at linear rates of 0.1, -3.5, 0.6, and-4.2 mm/a, respectively. ② The monthly non-seasonal T_WSC was significantly and negatively correlated with the Southern Oscillation Index, with a significance level of α < 0.01. ③ Climate variability played a leading role in the T_WSC variations, and the contribution rates of reservoir operations to the monthly mean T_WSC were negatively correlated with those of climate variability, and lastly. ④ Following the construction of the Three Gorges Reservoir, the influence of reservoir regulation on the monthly mean T_WSC was significantly enhanced; it presented seasonal variations in which T_WSC was reduced by reservoir operations during January—May and enhanced during July—December. This study developed a useful research framework for attribution analyses on T_WSC, and the results can provide decision support for water resource planning and management in the Yangtze River basin.