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Two main operating parameters (influent C/N ratio and electric current intensity) were examined for their impacts on the denitrifying bacterial community structure in an integrated system of three-dimensional biofilm-electrode reactor and sulfur autotrophic denitrification (3DBER-SAD). It was found that genus β-proteobacteria played a leading role under different operating conditions. The influent C/N ratio illustrated a great impact on denitrifying bacteria diversity. When the C/N ratio decreased from 1.07 to 0.36, the Shannon-Wiener index and Simpson index increased from 2.44 to 2.71 and from 0.89 to 0.92, respectively, while the proportion of heterotrophic denitrifying bacteria Thauera decreased from 61.4 to 21.1%, and the sulfur autotrophic denitrifying bacteria (e.g., genus Sulfuricella and Thiobacillus denitrificans) increased from 3.5 to 19.3%. In terms of the impact of electric current intensity, the Shannon-Wiener index and Simpson index decreased from 2.71 to 2.63 and from 0.92 to 0.90, respectively, as the current intensity increased from 60 to 400 mA.