CW-Fe allowed a high-performance of NO3‒-N removal at the COD/N ratio of 0. Higher COD/N resulted in lower chem-denitrification and higher bio-denitrification. The application of s-Fe0 contributed to TIN removal in wetland mesocosm. s-Fe0 changed the main denitrifiers in wetland mesocosm. Sponge iron (s-Fe0) is a porous metal with the potential to be an electron donor for denitrification. This study aims to evaluate the feasibility of using s-Fe0 as the substrate of wetland mesocosms. Here, wetland mesocosms with the addition of s-Fe0 particles (CW-Fe) and a blank control group (CW-CK) were established. The NO3‒-N reduction property and water quality parameters (pH, DO, and ORP) were examined at three COD/N ratios (0, 5, and 10). Results showed that the NO3‒-N removal efficiencies were significantly increased by 6.6 to 58.9% in the presence of s-Fe0. NH4+-N was mainly produced by chemical denitrification, and approximately 50% of the NO3‒-N was reduced to NH4+-N, at the COD/ratio of 0. An increase of the influent COD/N ratio resulted in lower chemical denitrification and higher bio-denitrification. Although chemical denitrification mediated by s-Fe0 led to an accumulation of NH4+-N at COD/N ratios of 0 and 5, the TIN removal efficiencies increased by 4.5%‒12.4%. Moreover, the effluent pH, DO, and ORP values showed a significant negative correlation with total Fe and Fe (II) (P<0.01). High-throughput sequencing analysis indicated that Trichococcus (77.2%) was the most abundant microorganism in the CW-Fe mesocosm, while Thauera, Zoogloea, and Herbaspirillum were the primary denitrifying bacteria. The denitrifiers, Simplicispira, Dechloromonas, and Denitratisoma, were the dominant bacteria for CW-CK. This study provides a valuable method and an improved understanding of NO3‒-N reduction characteristics of s-Fe0 in a wetland mesocosm.