Polyfluoroalkyl substance (PFAS) pose a threat to the aquatic environment due to their environmental persistence. The removal of PFAS using constructed wetlands (CWs) has received interest, but the adsorption saturation and limited removal capacity of the substrate is frequently challenging. To enhance the microbial degradation and performance of the substrate, different configurations of iron minerals were used as substrate to remove perfluorooctane sulphonic acid (PFOS) and perfluorooctanoic acid (PFOA) from CWs. The addition of iron minerals resulted in elimination of 57.2% and 63.9% of PFOS and PFOA in the effluent, respectively, which were 35.0% and 36.8% higher than that of control. Moreover, up to 85.4%, 86%, and 85.1% of NH4+, NO3-, and phosphorus, respectively, was removed using iron minerals. The enhanced electron transfer in iron mineral-based CWs was confirmed by a 61.2% increase in cytochrome C reductase content and an increased Fe(III)/Fe(II) ratio. Microbial analysis showed that the proportions of microbes with PFAS removal capacity (e.g. Burkholderiae and Pseudomonas), and the key pathways of the TCA cycle and glycolysis were increased in iron mineral-based CW. Based on these findings, we conclude that supplementation with iron mineral could enhance PFOA and PFOS removal in CWs.