The Three Gorges Dam has significantly altered ecological and environmental conditions within the reservoir region, but how these changes affect bacterioplankton structure and function is unknown. Here, three widely accepted metagenomic tools were employed to study the impact of damming on the bacterioplankton community in the Xiangxi River. Our results indicated that bacterioplankton communities were both taxonomically and functionally different between backwater and riverine sites, which represent communities with and without direct dam effects, respectively. There were many more nitrogen cycling Betaproteobacteria (e.g., Limnohabitans), and a higher abundance of functional genes and KEGG orthology (KO) groups involved in nitrogen cycling in the riverine sites, suggesting a higher level of bacterial activity involved in generating more nitrogenous nutrients for the growth of phytoplankton. Additionally, the KO categories involved in carbon and sulfur metabolism, as well as most of the detected functional genes also showed clear backwater and riverine patterns. As expected, these diversity patterns all significantly correlated with environmental characteristics, confirming that the bacterioplankton communities in the Xiangxi River were really affected by environmental changes from the Three Gorges Dam. This study provides a first comparative metagenomic insight for evaluating the impacts of the large dam on microbial function.