Anoxic thiosulfate (S2O3 2−) oxidation using autotrophic denitrification by a mixed culture of nitrate reducing, sulfur oxidizing bacteria (NR-SOB) was studied in a fluidized bed reactor (FBR). The long-term performance of the FBR was evaluated for 306 days at three nitrogen-to-sulfur (N/S) molar ratios (0.5, 0.3 and 0.1) and a hydraulic retention time (HRT) of 5 h. S2O3 2− removal efficiencies >99% were obtained at a N/S ratio of 0.5 and a S2O3 2− and nitrate (NO3 −) loading rate of 820 (±84) mg S-S2O3 2− L−1 d−1 and 173 (±10) mg N-NO3 − L−1 d−1, respectively. The S2O3 2− removal efficiency decreased to 76% and 26% at N/S ratios of 0.3 and 0.1, respectively, and recovered to 80% within 3 days after increasing the N/S ratio from 0.1 back to 0.5. The highest observed half-saturation (Ks) and inhibition (KI) constants of the biofilm-grown NR-SOB obtained from batch cultivations were 172 and 800 mg S-S2O3 2− L−1, respectively. Thiobacilus denitrificans was the dominant microorganism in the FBR. Artificial neural network modeling successfully predicted S2O3 2− and NO3 − removal efficiencies and SO4 2− production in the FBR. Additionally, results from the sensitivity analysis showed that the effluent pH was the most influential parameter affecting the S2O3 2− removal efficiency.