To explore whether and how anthropogenic activities related to surface water regulation (i.e., dam construction) disturb soil ecosystems in the surrounding uplands, a long-term monitoring program was conducted from 1998 to 2017 in the Three Gorges Reservoir Region, China. The Three Gorges Dam (TGD) is the largest hydraulic engineering project in the world. We present a direct, ecosystem-scale demonstration of changes in the soil organic carbon (SOC) content in the TGD watershed before and after the surface water was reshaped. The average SOC content decreased from 12.9 to 9.5 g/kg between 2004 and 2012 and then recovered to 13.8 g/kg in 2017. Dynamics of SOC were partly attributed to shifts in the composition of soil microbial communities responsible for carbon biogeochemistry. The shifts in microbial taxa were associated with the changed microclimate affected by the TGD as well as global and regional climate variability. The microclimate, soil microorganisms, and plant organic carbon input explained 40.2% of the variation in the SOC content. This study revealed that dam construction was an important and indirect driver for the SOC turnover, and the subsequent effects on the upland soil ecosystem must be considered when large-scale disturbance activities (such as dam construction) are conducted in the future.