•DOM chemistry was investigated in the Three Gorges Reservoir•Higher terrestrial input, molecular and isomeric complexity of DOM were detected in storage period•Hydrological management of reservoirs shifts the DOM chemistry•Hydrological management would affect DOM mineralization and CO2 emission in young reservoirs Reservoirs are well known as a far-reaching human modification on the functions of natural river networks. However, changes in the chemistry and reactivity of dissolved organic matter (DOM) responding to hydrological management for water retention structures, and its influence on the river carbon cycle, remain poorly understood. Here we show that hydrological management does shape the molecular composition of DOM in the world's largest Three Gorges Reservoir, as revealed by optical spectroscopy and ultrahigh-resolution mass spectrometry. Relatively higher terrestrial input, molecular complexity, isomeric complexity, and environmental stability of DOM were observed during the storage period, whereas the inverse occurred during the drainage period. The results demonstrate that the hydrodynamic processes, which are mainly controlled by water intrusion from mainstream to tributaries, are likely the underlying mechanism controlling DOM chemistry. Integrated with observations from worldwide river reservoirs, the DOM degradation experiments suggest that reservoir hydrological management would enhance DOM mineralization, thereby increase CO2 emission and change the river carbon cycle. Display omitted