Azo dye is the most versatile class of dyes used in the textile industry. Although the sulfidogenic process shows superiority in the removal of azo dye, the role of biogenic sulfide produced by sulfate-reducing bacteria (SRB) in the decolorization of azo dye is unclear. This study explored the mechanism of biogenic sulfide for removal of a model azo dye (Direct Red 81 (DR 81)) through biotic and abiotic batch tests with analysis of intermediates of the azo dye degradation. The results showed that biogenic sulfide produced from sulfate reduction directly cleaved two groups of azo bond (-N[dbnd]N-), thereby achieving decolorization. Moreover, the decolorization rate was enhanced by nearly 3-fold (up to 42 ± 1 mg/L-hr; removal efficiency > 99%) by adding an external carbon source or elevating the initial azo dye concentration. This study showed that biogenic sulfide plays an essential role in azo dye decolorization and provides a new avenue for the potential application of biogenic sulfide from the sulfidogenic system for the treatment of azo dye-laden wastewater.