Paddy soil was collected from Southern China and used as an inoculum in a microcosm experiment. The microcosm experiment was used to study the process of autotrophic nitrate reduction coupled with As(III) oxidation and its microbial community structure in the flooded paddy field. As(III) was completely oxidized in As(III)+NO₃^- treatment, while no As(III) oxidation was observed in the As(III) amendment and the sterilization treatment (Sterilized soil+As(III)+NO₃^-). Meanwhile, a small amount of NO₃^- reduction was observed in As(III)+NO₃^- amendment and no NO₃^- reduction was observed in NO₃^- amendment. 16S rRNA-based high-throughput sequencing was used to investigate the microbial community structure in Soil+As(III)+NO₃^- treatment. Diversity of soil bacterial community was decreased and its Shannon index was 8.19in Soil+As(III)+NO₃^- treatment. The dominant bacteria at the phyla level were Proteobacteria (33%), Chloroflexi (11%), and Planctomycetes (12%), and the dominant genus were Gemmatimonas (7.4%), with few Singulisphaera、Thermomonas、Bacillus in Soil+As(III)+NO₃^- treatment. In summary, the addition of NO₃^- can promote the autotrophic As(III) oxidation and influences the microbial community composition in flooded paddy soil.