The construction of dams and subsequent water level fluctuations significantly alters the environmental conditions of reservoir ecosystems, affecting the metabolic pathways of CO2 fixing microorganisms (CFMs) and carbon storage in drawdown areas. We investigated the response of soil bacterial communities and carbon fixation pathways to periodic flooding and drying at different elevations by collecting surface soil samples (0–10 cm) in the drawdown area of the Three Gorges Reservoir. The results show that periodic flooding and drying increased the complexity of bacterial co‐occurrence networks. We identified the reductive citrate cycle (rTCA cycle), dicarboxylate‐hydroxybutyrate cycle (DC/4‐HB cycle), 3‐Hydroxypropionate cycle (3‐HP cycle) and reductive pentose phosphate cycle (Calvin cycle) as the main carbon fixation pathways. Notably, as the inundation duration increased, the abundance of the Calvin cycle and its key gene (cbbL) gradually decreased. We also found that soil moisture, pH and the different organic carbon components affected the composition of bacterial communities and the abundance of carbon fixation pathways. These findings elucidate evolutionary trends bacterial communities and the impacts of water level fluctuations on microbial‐mediated carbon cycling processes because of dam construction.