Methane-driven hexavalent chromium (Cr(VI)) reduction in a microbial fuel cell (MFC) has attracted much attention. However, whether the presence of sulfate (SO42−) affects the reduction of Cr(VI) is still lacking in systematic studies. This study involved constructing a MFC-granular sludge (MFC-GS) coupling system with dissolved methane (CH4) was used as the electron donor to investigate the effect of SO42− on Cr(VI) bioreduction, sludge characteristic, and functional metabolic mechanisms. When the SO42− concentration was 10 mg/L, the average removal rate of Cr(VI) in the anaerobic stage decreased to the lowest value (22.25 ± 2.06%). Adding 10 mg/L SO42− obviously inhibited the electrochemical performance of the system. Increasing SO42− concentration weakened the fluorescence peaks of tryptophan and aromatic proteins in the extracellular polymeric substance of sludge. Under the influence of SO42−, Methanothrix_soehngenii decreased from 14.44% to 5.89%. The relative abundance of methane metabolic was down-regulated from 1.47% to 0.98%, while the sulfur metabolic was up-regulated from 0.09% to 0.21% when SO42− was added. These findings provided some reference for the treatment of wastewater containing Cr(VI) and SO42− complex pollutants in the MFC-GS coupling system.