The present work investigates the impacts and mechanisms associated with natural organic matter (NOM) in the Fe0 treatment system of Cu2+ and Zn2+ under roof runoff conditions. The NOM in runoff waters was characterized using XAD-4/8 adsorption resins, copper complexation, acidic capacity and liquid chromatography with online carbon detection. Batch kinetic experiments and flow-through configurations were performed and the results of metal removal were elucidated taking into account the characteristics of NOM. Based on the findings, it is shown that NOM influences the removal of metals through several complex pathways. At an un-favored condition for adsorption of metals, i.e., on iron corrosion products, at pH<<pH(PZC), the previously adsorbed NOM noticeably enhances the overall removal of metals by forming of an oxide-NOM-metal ternary complex. In a more typical condition for roof runoff (i.e., pH-pHPZC), metal-ligand complexes in the bulk water as well as a competitive adsorption between them play important roles on the removal processes. A significant decrease in the treatment efficiency observed during flow-through configuration suggests a hinderance effect of NOM on corrosion of iron by surface coverage. The NOM fraction that mainly interacts with the removal of metals is a larger size hydrophobic fraction with high aromaticity. In the practical operation, continuous input of NOM will lessen the effectiveness of the treatment barrier. However, fate and toxicity of the metals in natural environments has to be assessed also by complexation capacity of NOM.