The searches for suitable substrates with high capacity for phosphorus (P) removal and promoting denitrification for enhancing nitrogen (N) removal have been a key work in constructed wetlands (CWs) research in the past several decades. But few substrates enhancing simultaneous long-term N and P removal in CWs have been found before. In this study, two subsurface flow pilot-scale wetlands using natural pyrite and limestone as substrates were constructed. After 3 year of operation, we found that pyrite had no negative effects on growth of reeds, removals of chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N), but enhanced long-term total nitrogen (TN) and total phosphorus (TP) removals in constructed wetland. In the three years, the average TP and TN removals of pyrite constructed wetland (PCW) were 87.7 ± 14.2% with 0.25 ± 0.20 mg/L of average effluent TP and 69.4 ± 21.4% with 4.0 ± 3.2 mg/L of average effluent TN, respectively. The main P form in the PCW was (Fe + Al)-bound P. The mechanisms of the PCW with enhanced simultaneous long-term N and P removals were anaerobic and aerobic oxidations of pyrite. The main bacteria were Anaeromyxobacter (4.9%), Ramlibacter (4.8%), Defluviicoccus (4.2%), Azoarcus (3.7%), Geobacter (3.4%), and they were highly related to anaerobic and aerobic oxidation of pyrite in the PCW. Display omitted •Pyrite can enhance long-term TN and TP removal in constructed wetland.•Pyrite had no negative effects on growth of reeds, removals of COD and NH4+-N.•The main retained P was in form of (Fe + Al)-bound P in pyrite constructed wetland.•Anaerobic and aerobic oxidations of pyrite improved TN and TP removal in PCW.