Sulfadiazine (SDZ) is a high priority sulfonamide antibiotic and was always detected in environmental samples. This study explored the removal of SDZ in microbial fuel cells (MFCs), in terms of MFC operation, degradation products, reaction mechanism, SDZ biotoxicity removal, and the correlation between microbial community and SDZ removal. SDZ would greatly impact the activity of reactor microbes, and longtime acclimation is required for the biodegradation of SDZ in MFCs. After acclimation, 10 mg/L of SDZ could be removed within 48 h. Liquid chromatographic-mass spectroscopic analysis showed that SDZ could be degraded into 2-aminopyrimidine, 2-amino-4-hydroxypyrimidine and benzenesulfinic acid. Compared with published SDZ biodegradation mechanism, we found that the sulfanilamide part (p-Anilinesulfonic acid) of SDZ would be degraded into benzenesulfinic acid in the system. The effects of background constituents on SDZ biodegradation were explored, and co-existed humic acid (HA) and fulvic acid (FA) could accelerate the removal of SDZ in MFCs. After analyzing the reactor microbial community and the removal of SDZ at different operation cycles, it was found that the relative abundance of Methanocorpusculum, Mycobacterium, Clostridium, Thiobacillus, Enterobacter, Pseudomonas, and Stenotrophomonas was highly correlated with the removal of SDZ throughout the experiment.