Utilizing natural wetlands to remove phosphorus (P) from agricultural drainage is a feasible approach of protecting receiving waterways from eutrophication. However, few studies have been carried out about how these wetlands, which act as buffer zones of pollutant sinks, can be operated to achieve optimal pollutant removal and cost efficiency. In this study, cores of sediments and water were collected from a lacustrine wetland of Lake Xiaoxingkai region in Northeastern China, to produce a number of lab-scale wetland columns. Ex situ experiments, in a controlled environment, were conducted to study the effects of aeration, vegetation, and iron (Fe) input on the removal of total P (TP) and values of dissolved oxygen (DO) and pH of the water in these columns. The results demonstrated the links between Fe, P and DO levels. The planting of Glyceria spiculosa in the wetland columns was found to increase DO and pH values, whereas the Fe:P ratio was found to inversely correlate to the pH values. The TP removal was the highest in aerobic and planted columns. The pattern of temporal variation of TP removals matched first-order exponential growth model, except for under aerobic condition and with Fe:P ratio of 10:1. It was concluded that Fe introduced into a wetland by either surface runoff or agricultural drainage is beneficial for TP removal from the overlying water, especially during the growth season of wetland vegetation.