A novel membrane-bound hydrogen sulfide dehydrogenase was purified to homogeneity by a four-step procedure from Thiobacillus thioparus D6, an neutrophilic, obligately chemolithoautotrophic bacteria obtained from aerobic magnetic stabilized fluidized bed reactor of flue gas biodesulfurization system. The natural hydrogen sulfide dehydrogenase had a molecular mass of 95 kD and comprised two subunits named α and β with molecular masses of 42.6 kD and 51.3 kD determined by exclusion chromatography and SDS-PAGE. Spectral and pyridine hemochrome analysis revealed that the enzyme contained 1 mol flavin and 1 mol haem c per mol αβ hydrogen sulfide dehydrogenase, assumed a characteristics of the member of redox proteins. Biochemical determination and nonlinear regression analysis showed that the sulfide dehydrogenase catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 8.6 with a kcat of 32.4 s?1, a Km of 6.1 μmol/L for sulfide, and a Km of 2.5 μmol/L for cytochrome c. The yield of 1.9 mol of cytochrome c reduction per mole of sulfide suggested that the product was sulfur or polysulfide. The activity of the sulfide dehydrogenase was inhibited by sulfur and sulfite like that cyanide (100 μmol/L) inhibited sulfide dehydrogenase activity at pH 6.0 by 72%.