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Sulfur autotrophic denitrification has been proved feasible for nitrate removal from aquatic environments and it utilizes elemental sulfur as the electron donor. A maximum denitrification rate of 194.57 mg N/L·d was achieved with biogenic sulfur as electron donor in a mixed culture collected from sulfur packed bed reactors; this rate was considerably higher than that delivered by α-S 8 or μ-S in the same mixed culture. The elemental sulfur was also tested in the pure culture of Thiobacillus denitrificans , while a lower denitrification rate was noted than in the mixed culture, bio-S (4.86 mg N/L·d) again outperformed other two elemental sulfur's. X-ray absorption near edge structure spectra were collected to examine possible metabolic intermediates during the sulfur autotrophic denitrification process. The analysis revealed the existence of two major intermediates: DL-cysteine and L-cystine. They were found to not only provide electrons but also play a critical role in promoting the elemental sulfur-mediated sulfur autotrophic denitrification process. In general, we investigated the formation and enhancement effects of sulfur intermediates in the sulfur autotrophic denitrification process. Unlabelled Image • Three forms of sulfurs were used for So-based denitrification process. • Bio-S could achieve the best nitrogen removal performance. • XANES analysis revealed the existence of DL-cysteine and L-cystine in bio-S. • DL-cysteine and L-cystine were able to improve So-based denitrification process. [ABSTRACT FROM AUTHOR]