The　partial　denitrification/anammox　(PD/A)　process　is　receiving　increasing　attention　due　to　its　cost-effectiveness　advantages.　However,　effective　strategies　to　alleviate　organic　matter　inhibition　and　promote　anammox　activity　have　been　proven　to　be　a　big　challenge.　This　study　investigated　the　effects　of　three　types　of　iron　(nano　zero-valent　iron　(nZVI),　Fe(II),　and　Fe(III))　on　the　PD/A　process.　It　is　worth　noting　that　nZVI　of　5–50　mg/L　and　Fe(III)　of　5–120　mg/L　promoted　both　PD　and　anammox　activity.　Long-term　intermittent　addition　of　nZVI　(50　mg/L)　resulted　in　a　nitrogen　removal　efficiency　of　98.2%　in　the　mixotrophic　PD/A　system　driven　by　iron　and　organic　matter.　The　contribution　of　anammox　for　nitrogen　removal　reached　as　high　as　93.8%.　The　organic　carbon　demand　decreased　due　to　the　external　electron　donor　provided　by　nZVI　for　PD.　Multiple　Fe–N　metabolic　pathways,　primarily　involving　ammonia　oxidation　by　Fe(III)　and　nitrate　reduction　by　nZVI,　play　a　crucial　role　in　facilitating　nitrogen　transformation.　Conversely,　the　direct　addition　of　30–120　mg/L　Fe　(II)　resulted　in　a　significant　decrease　in　pH　to　below　5.0　and　severe　inhibition　of　PD　and　anammox　activity.　Following　prolonged　operation　in　the　presence　of　nZVI,　it　was　demonstrated　that　there　is　an　enhancing　effect　on　robust　nitrite　production　for　anammox.　This　was　accompanied　by　a　remarkable　up-regulation　of　genes　encoding　nitrate　reductase　and　iron-transporting　proteins　dominated　by　Thauera.　Overall,　this　study　has　provided　an　efficient　approach　for　advanced　nitrogen　removal　through　organic-　and　iron-driven　anammox　processes.　©　2024　Elsevier　Ltd