[Background] Anaerobic oxidation of methane (AOM) includes denitrification- dependent anaerobic methane oxidation and sulfate reduction-dependent anaerobic methane oxidation. At present, the excessive discharge of nitrogen and sulfur pollutants into water causes serious environmental pollution and ecological damage. [Objective] We used the AOM-microbial fuel cell (MFC) to study the coupling reaction mechanism of simultaneous nitrogen and sulfur removal and the microbial diversity changes during the reaction. [Methods] We constructed three MFCs (N-S-MFC, N-MFC, and S-MFC) with methane as the sole carbon source to explore the simultaneous nitrogen and sulfur removal performance. The 16S rRNA gene high-throughput sequencing was employed to analyze the microbial community structure. [Results] The removal rates of nitrate and sulfate in N-S-MFC were 90.91% and 18.46%, respectively. The relative abundance of microorganisms in the anode chamber increased, and the microorganisms involved in denitrification and sulfate reduction were enriched. Specifically, the relative abundance of common methanotrophs including Bacteroidota, Firmicutes and Desulfobacterota at the phylum level and Methylobacterium_Methylorubrum, Methylocaldums and Methylomonas at the genus level was increased. [Conclusion] N-S-MFC promotes nitrate reduction while having little effect on sulfate reduction. This study provides a theoretical basis for the application of methane MFC in simultaneous nitrogen and sulfur removal from wastewater.