Xiangxi River is the largest inflow tributary in the Three Gorges Reservoir area. Structural characteristics and influence factors of the microbial communities in sediments of Xiangxi River are not clear so far. This study investigated the main physical and chemical parameters of the overlying water (i.e., surface, middle and bottom layers) and sediments of the Xiangxi River. 16S rDNA high-throughput sequencing was used to reveal the characteristics of microbial community structures in the sediments. In addition, the relationship between bacteria and physicochemical parameters were analyzed statistically. Chlorophyll.a (Chl.a), concentrations of ammonia nitrogen (NH₃-N) in the overlying water increased gradually from the downstream (2.04 mg/L) to the upstream (3.14 mg/L). The concentrations of phosphate (PO^3-₄) in overlying water fluctuated between 0.14 and 0.18 mg/L at different sampling sites. In addition, the concentrations of NH₃-N and PO^3-₄ in the pore-water at the same site were significantly higher than those in the overlying water. Pore-water NH₃-N and PO^3-₄ concentrations at 10-15 cm depth were 3.4 and 1.7 times higher than those at 0-5 cm depth in sediments, respectively. Higher contents of Fe (Ⅱ), Fe (Ⅲ), acid volatile sulfide and chromium reducing sulfide were observed in the upstream sampling sites and bottom layers of the sediments. The microbial diversity and richness in the sediments of midstream were higher compared to the sediments of downstream and upstream. Proteobacteria was the dominant phylum in the surface sediments, and Sphingomonas was the main genus. The spatial distributions of microorganisms for different metabolic activities varied. Typical bacteria with the ability to degrade polycyclic aromatic hydrocarbons, such as Novosphingobium and Terrimonas, were mainly observed in the sediments of downstream sites with percentage reaching 5.8% and 5.4%, respectively. The percentages of Nitrospira and Flavobacterium were highest in the midstream. The iron-oxidizing bacteria (i.e., Aquabacterium) were rich in the upstream sites. Redundancy analysis (RDA) showed that total organic carbon (TOC), sulfate (SO^2-₄), Fe (Ⅱ), NH₃-N and total phosphorus (TP) were the main environmental factors shaping the microbial community structures in sediments of Xiangxi River. This study will help to further understand the impacts of surrounding environment and human activities on the stability of water quality and the microorganisms in the sediments of Xiangxi River, and provide a reference for the improvement and management of the water environment in the Three Gorges Reservoir area.