This study investigated the feasibility of a new biological nitrogen removal process that integrates sulfur-driven autotrophic denitratation (NO₃ ⁻→NO₂ ⁻) and anaerobic ammonium oxidation (Anammox) for simultaneous removal of nitrate and ammonium from industrial wastewater. The proposed sulfur(thiosulfate)-driven denitratation and Anammox process was developed in two phases: First, the thiosulfate-driven denitratation was established in the UASB inoculated with activated sludge and fed with ammonium, nitrate and thiosulfate for 52 days until the nitrite level in the effluent reached 32.1 mg N/L. Second, enriched Anammox biomass was introduced to the UASB to develop the integrated thiosulfate-driven denitratation and Anammox (TDDA) bioprocess (53–212 d). Results showed that nitrate and ammonium could be efficiently removed from synthetic wastewater by the integrated TDDA system at a total nitrogen (TN) removal efficiency of 82.5 ± 1.8% with an influent NH₄ ⁺-N of 101.2 ± 2.2 mgN/L, NO₃ ⁻-N of 101.1 ± 1.5 mgN/L and thiosulfate of 202.5 ± 3.2 mg S/L. It was estimated that Anammox and autotrophic denitritation (NO₂ ⁻→N₂) contributed to about 90% and 10% of the TN removal respectively at stable operation. The established TDDA system was further supported by high-throughput sequencing analysis that sulfur-oxidizing bacteria (e.g., Thiobacillus and Sulfurimonas) coexisted with Anammox bacteria (e.g., Ca. Kuenenia and Ca. Anammoxoglobus) in this syntrophic biocenosis. Additionally, batch experiments were conducted to reveal the kinetic rates and to reconcile the stoichiometry of the electron donor/acceptor couples of the TDDA process. The results unraveled the mechanisms in the new bioprocess: i) sulfite and elemental sulfur (S⁰) were initially generated from branched thiosulfate; ii) oxidation of sulfite and elemental sulfur coupled with fast and slow denitratation; iii) nitrite produced from denitratation together with ammonium were effectively converted to dinitrogen gas via Anammox.