Abstract:[Objective] The purpose of this paper is to explore the distribution of bacterial communities along different water depths in a pit lake affected by acid mine drainage (AMD) and its relationship with environmental factors. [Methods] The 16S rRNA gene high-throughput sequencing technology was used to investigate the bacterial communities along different water depths at 6 sampling sites in the lake. The physicochemical indexes of water quality were measured at the same time. The relationship between bacterial distribution and water physicochemical indexes was analyzed by statistical software. [Results] The physicochemical properties and bacterial distribution showed obvious stratification. Dissolved oxygen (DO) decreased while pH and concentrations of metal ions increased from top to bottom. Meanwhile, the bacterial diversity and the abundance of some species increased from top to bottom. Proteobacteria (Alpha, Gammaproteobacteria) and Acidobacteria were dominated in the surface water, while Firmicutes, Acidobacteria,Actinobacteria, Gammaproteobacteria and Patescibacteria were rich in the middle- and deep-layer water. The total nitrogen (TN), DO, oxidation-reduction potential (ORP), pH, Fe, Mn, Al and Zn were significantly correlated with the abundance of acidophilic bacteria, which were the main factors influencing the spatial distribution of bacteria. [Conclusion] The distribution of bacteria in the pit lake affected by AMD showed an obvious vertical pattern, which was caused by the joint action of various environmental factors. This study has reference value for understanding the microbial distribution in the AMD-influencing pit lake and in situ bioremediation of AMD.