A pilot-scale step-feed A/O system was used to treat low C/N municipal wastewater. To obtain high quality simultaneous nitrogen and phosphorus removal, a modified step-feed A/O system was developed and the nitrogen and phosphorus removal efficiency was evaluated between the original and modified system. The result showed that TN removal efficiency was 66.52% and TP removal efficiency was only 29.24% before the modified configuration was applied. When the modified system was adopted, the satisfied simultaneous nitrogen and phosphorus removal was achieved. TP removal efficiency increased to 89.81% and TN removal efficiency was 73.61% when the optimal feeding ratio (0.45:0.35:0.20) was applied. Compared to the original configuration, TN removal efficiency increased about 7.09% due to the enhanced denitrifying phosphorus removal, which saved the carbon source for denitrification. To evaluate the selection and dominance of polyphosphate-accumulating organisms (PAOs) and denitrifying polyphosphate-accumulating organisms (DNPAOs), batch test was explored to examine the anaerobic phosphorus release, aerobic and anoxic phosphorus uptake. The result showed that both PAOs and DNPAOs were accumulated gradually when the modified system was applied. The maximal aerobic P uptake rate [P/(MLSS x t)] was increased from 2.34 mg/(g x h) to 10.67 mg/(g x h) and the anoxic P uptake rate was increased from 0.33 mg/(g x h) to 2.81 mg/(g x h) when the modified system was operated.