Abstract: Sulfur-autotrophic reduction of nitrate from water by up-flow packed bed reactor was investigated to explore the generation regularity of sulfur by-products. The effect of hydraulic retention time (HRT) on NO₃^--N removal was examed. The NO₃^--N removal kinetics was fitted with the zero-order and half-order kinetics models. The generation trend of sulfur by-products was analyzed by experimental results and theoretical calculation. High-throughput sequencing was used to reveal the microbial community spatial distribution. The results showed that:(1) The removal efficiency was more than 98% when NO₃^--N was at (30.45±0.38)mg/L in the influent and HRT shortened from 4 h to 1 h. Half-order kinetics model fits the experimental data well with 1/2K_1/2V at 5.69 mg^1/2/(L^1/2·h); (2) The SO₄^2- production in the effluent was in line with the theoretical value. Trace of S^2- was accumulated in the reactor and further decreased in the effluent (< 0.5 mg/L); (3) The shortened HRT changed the α-biodiversity of the microbial community. Proteobacteria was the dominant bacteria, and its proportion in each stage was greater than 59%. Sulfurimonas was the most common denitrifying bacterium and its abundance at 1 h was 36%, then became the dominant species. The increase of sulfate-reducing bacterium (Desulfurella) abundance was consistent with the accumulation of S^2-. The SO₄^2- concentration in the effluent was in good agreement with the theoretical value, and the S^2- concentration displayed the tendency of rising up with the reactor height and then decreasing in the effluent. The distribution of microbial community structure was related to reactor height.