Sulfide oxidation is coupled with electricity generation in a sulfide-fed microbial fuel cell (MFC). This study demonstrated that both electrochemical reactions and microbial catalysis were involved in such a complex sulfide oxidation process in the anode of an MFC. The microbe-assisted sulfide oxidation generated a higher persistent current density than the sulfide oxidation via single electrochemical reactions only. Three valence states of S (-II), S (0), and S (+VI) were discovered from the sulfide oxidation, and So, Sx(2-), S4O6(2-), S2O3(2-), and SO4(2-) were detected as the intermediates. Based on the sulfur speciation and microbial community analysis, the sulfide oxidation pathways in the MFC were proposed. The oxidation of sulfide to So/Sx(2-) and further to S4O6(2-)/S2O3(2-) occurred spontaneously as electrochemical reactions, and electricity was generated. The formation of So/Sx(2-) and S2O3(2-) was accelerated by the bacteria in the MFC anode, and SO4(2-) was generated because of a microbial catalysis. The microbe-assisted production of S2O3(2-) and SO4(2-) resulted in a persistent current from the MFC.