Abstract Thiosulfate is frequently used as an energy source and electron donor in autotrophic denitrification (AD) for removing nitrate from wastewater. However, transforming pathways of S2O32− in this process is unclear. Herein, the aim of this study is to explore possible transforming pathways of sulfur compounds in thiosulfate-based AD process. After measuring the variation of NO3−, NO2−, and various sulfur compounds such as S0, SO42−, S2O32−, acid volatile sulfide (AVS), and S2− in the presence and absence of S2O32−, the variation process of S2O32− and the contribution of various sulfur compounds were analyzed. The results indicated that S0, AVS, and S2− were the intermediate products when S2O32− was applied as an electron donor. All S2O32−, S0, AVS, and S2− could act as electron donors in the nitrate removal process with the final products of SO42−. The utilization priority of these four sulfur sources was presumed in the following order: S2− > S2O32− > AVS ≈ S0. Furthermore, sulfur transformation and balance in nitrate removal process was also investigated. This suggests the transforming pathways of sulfur compounds in denitrification process. Nitrogen removal and sulfur conversion process are dependent on the presence of microorganisms in the sludge.