Conventional　autotrophic　nitrification　process　is　difficult　to　treat　high-temperature　wastewater　with　high-strength　ammonia.　In　this　study,　a　high-temperature　(50　°C)　biofilm　system　based　on　heterotrophic　nitrification　and　aerobic　denitrification　(HNAD)　was　established.　The　results　showed　that　the　HNAD　process　was　high　temperature　resistant,　and　the　nitrogen　removal　performance,　pathway　and　microbial　mechanism　varied　remarkably　at　different　temperatures.　The　high-temperature　system　showed　excellent　nitrogen　and　COD　removal　capacities　at　50　°C.　Ammonia　oxidation　was　mainly　undertaken　by　heterotrophic　nitrification,　while　anoxic　and　aerobic　pathways　worked　in　concert　for　denitrification.　High-throughput　sequencing　indicated　that　heterotrophic　nitrifying　bacteria　(8.58%)　and　denitrifying　bacteria　(52.88%)　were　dominant　at　50　°C.　Metagenomic　analysis　further　suggested　that　the　carbon　metabolism　was　up-regulated　in　response　to　the　increasing　temperature,　so　more　energy　was　generated,　thereby　promoting　the　HNAD-related　nitrogen　removal　pathways.　The　study　revealed　the　microbial　mechanism　of　HNAD　at　high　temperature　and　provided　new　insights　into　high-temperature　biological　nitrogen　removal.　©　2021　Elsevier　Ltd