The loss of CO2 to the atmosphere from inland waters is an important part of the global carbon cycle. The Three Gorges Dam is the largest hydraulic project in the world and has consequently been widely studied. Here, we made spatially and temporally comprehensive measurements of partial pressure of CO2 (pCO2) variability along the Three Gorges Dam system. The pCO2 ranged from 619 to 2383 μatm for the collected samples, and were supersaturated relative to atmospheric CO2. At the station near the upstream part of the reservoir, the pCO2 at high-flow was much lower than that at low-flow. In contrast, pCO2 at high-flow is much higher than that in the low-flow for the waters in front of the dam and after the dam. Rates of organic matter mineralization increased at high-flow, which produced increased pCO2 in the surface water of the reservoir area. Mineralization of organic carbon should be responsible for the 13 C-depleted of riverine DIC. Organic carbon mineralization is sensitive to temperature variability, and temperature is expected to be an important driver of the dissolved CO2 over-saturation. The construction of Three Gorges Reservoir increased the water transit time and accelerated the organic carbon mineralization in Three Gorges Reservoir. The results suggest that carbon cycling changes markedly in large rivers that have been impounded.