Abstract: Four sets of bench-scale anoxic/oxic biofilters (A/O-BF) were set up to study the influence of hydraulic retention time (HRT) on the nitrogen removal from simulated low carbon-to-nitrogen (C/N) ratio rural domestic sewage. Combined with the high-throughput sequencing method, the characteristics of nitrogen removal efficiency and microbial communities were extensively investigated at different HRTs, and the best HRT for the anoxic and oxic column of A/O-BF was recommended. The results were as follows: (1) When the HRT of anoxic column maintained at 6 h, the average NH₄⁺-N and TN removal efficiency of A/O-BF was 99% and 75% at 9 h and 10 h HRT in the oxic column, which was significantly higher than those at 7 h (93% and 66%) and 8 h (96% and 70%) HRT. (2) When the HRT of the oxic column was 9 h, the TN removal efficiency of A/O-BF was 78% at 6 h HRT in the anoxic column, which was significantly higher than that at 4 h (59%) and slightly higher than that at 5 h (74%) and 7 h (75%) HRT. (3) The total proportion of Nitrosomonas and Nitrospira in the oxic column at 9 h (11.42%) HRT was significantly higher than that at 7 h (4.61%), 8 h (7.39%) and 10 h (7.64%) HRT. The proportion of the major denitrifying bacteria, i.e. Denitratisoma in the anoxic column at 6 h (5.83%) HRT was significantly higher than that at 4 h (0.81%), 5 h (2.90%) and 7 h (3.27%) HRT. (4) The nitrogen removal efficiency and microbial community distribution of A/O-BF were influenced by the HRT, and 9 h and 6 h were the optimum HRT for the oxic and anoxic column of A/O-BF during the treatment of rural domestic sewage with low C/N ratio.