Background: Background: Increasing nitrate concentrations in groundwater resources is considered a common environmental and public health problem worldwide. In this research, an autotrophic up-flow bioreactor with pumice as media was used to study the effects of the sulfur-to-nitrogen (S/N) ratio and empty bed contact time (EBCT) on nitrate removal efficiency and byproducts. Methods: Experiments were carried out in a 3.47 L up-flow, fixed-bed reactor with 3 sampling ports. To evaluate the overall impact of S/N ratio and EBCT on the performance of the bioreactor, several phases with different S/N ratios and EBCTs were applied. Results: At a constant S/N ratio of 3.85 g/g, as EBCT decreased from 24 hours to 2 hours, the nitrate removal efficiency decreased from 98% to 64%. On the other hand, at the desired EBCT of 4 hr, as S/N ratio decreased from 3.85 to 1.51 g/g, nitrate removal efficiency was reduced from 85% to 32%. Changing the EBCT and S/N ratio also affected the effluent nitrite and sulfate concentrations as byproducts. At the S/N ratio of 3.85 g/g and EBCT of 24 hours, effluent nitrite and sulfate concentrations were 0.1 mg NO 2-N/L and 463 mg SO 4 2-/L, respectively. Decreasing the S/N ratio to 1.51 g/g and the EBCT to 4 hours caused drastic changes in effluent nitrite and sulfate concentrations. Conclusion: The results indicated that the autotrophic denitrification with thiosulfate as electron donor and pumice as media was feasible and applicable for nitrate contaminated groundwater.