•Mixotrophic denitrification (C/N = 3) was achieved in the pyrite-based VFCW.•The highest TN and TP removal rate were achieved with the pyrite addition (100%).•The mixed addition of pyrite was better for N and P removal.•The autotrophic and heterotrophic bacteria were significantly promoted by pyrite. Deep nitrogen removal from low-carbon wastewater is a pressing water treatment challenge as of yet. Eight sets of vertical-flow constructed wetland (VFCW) intensified by pyrite were designed and applied to treat with low C/N ratio wastewater in this research. The results showed that the addition of pyrite (100% added) significantly promoted TN removal with an efficiency higher than 27.05% under low C/N ratio conditions, indicating that mixotrophic denitrification was achieved in VFCW. Microbial analysis showed that the community structure and diversity of microorganisms were changed significantly, and the growth of autotrophic (Thiobacillus) and heterotrophic bacteria (Thauera) concomitantly enhanced. It is recommended that the addition amount of pyrite is 75% of the wetland volume, meantime, mixing evenly with 25% high porosity substrate (such as activated carbon, volcanic stone, etc.), which could enhance the effective adhesion of microorganisms and their contact area with pyrite, ultimately improve the denitrification capacity of the VFCW.