The　slow　growth　and　difficulty　in　cultivating　anammox　bacteria　limit　the　rapid　start-up　of　anammox　process　and　effective　microbial　enrichment.　In　this　study,　microbial　electrolysis　cell　(MEC)　was　coupled　with　anammox　to　investigate　the　effects　of　different　applying　voltage　methods　on　substrate　removal　efficiency　and　rates,　microbial　community　structure,　anammox　metabolism　and　metabolic　pathways.　The　results　showed　that　applying　voltage　not　only　improved　NH4+-N　removal　efficiency　and　removal　rates,　but　also　promoted　electron　transfer　efficiency,　key　enzyme　activity　and　extracellular　polymeric　substances　(EPS)　secretion　in　the　systems.　Step-up　voltage　was　more　conducive　to　the　growth　of　Candidatus_Kuenenia　in　the　cathode,　which　promoted　the　rapid　start-up　of　anammox　and　treating　wastewater　with　low　ammonia　concentration.　The　main　metabolic　pathway　in　step-up　voltage　operation　was　hydrazine　to　nitrogen,　while　in　constant　voltage　operation　was　hydroxylamine　oxidation　pathway.　These　findings　provided　a　new　insight　into　the　enhancement　and　operation　of　anammox　system.　©　2023　Elsevier　Ltd