【Objective】The concentration and speciation of arsenic in groundwater are significantly influenced and regulated by microbial activities. The Kuitun River Basin is a typical high-arsenic area, yet research on the metabolic characteristics of arsenic-metabolizing microorganisms in groundwater from this region remains scarce.【Methods】In this study, 11 groundwater samples were collected from the Kuitun River Basin for metagenomic sequencing. The chemical characteristics of high- and low-arsenic groundwater were analyzed, along with the associations between the community structure and functional genes of arsenic-metabolizing microorganisms and environmental factors.【Result】The results indicated that the dominant bacterial phyla in the groundwater of the study area were Proteobacteria, Actinobacteria, and Planctomycetota. At the genus level, the microbial communities in low-arsenic samples exhibited significant species richness and diversity advantages. The abundances of Nitrosomonas, Thiobacillus, and Sulfuritalea were significantly positively correlated with total arsenic. Microorganisms involved in the nitrogen cycle can indirectly affect the arsenic metabolism process. A total of 12 functional genes related to arsenic methylation, arsenite (As(III)) oxidation, arsenate (As(V)) reduction, and arsenic transport were annotated in this study. Total arsenic (As) was significantly negatively correlated with arsC1 (arsenate reductase gene) and arsH (organic arsenical oxidase gene), and significantly positively correlated with AS3MT (arsenic methyltransferase gene).【Conclusion】Arsenic concentrations can inhibit the abundance and diversity of arsenic-metabolizing microbial communities and functional genes, exerting selective pressure on the microbial community structure and enriching arsenic-resistant microorganisms in high-arsenic environments. Multiple arsenic metabolism pathways exist among groundwater microorganisms in the study area, including the As(V) efflux system composed of ArsJ (organic arsenical efflux permease) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Arsenic methylation is the primary pathway of microbial arsenic metabolism in high-arsenic groundwater in this basin. The relationship between arsenic-metabolizing microbial communities and functional genes and total arsenic is regulated and influenced by multiple environmental factors, rather than by a single factor. By screening microbial populations with arsenic metabolism capabilities and their key functional genes, this study not only deepened our understanding of arsenic metabolism mechanisms but also provided new strategies for bioremediation technologies for arsenic contamination.