The mechanisms behind nitrogen removal by the submersed macrophyte-biofilm complex in wetlands remain to be fully elucidated. This study investigated the role of Myriophyllum spicatum and the biofilm on their leaves in nitrogen removal in mesocosm experiments. N-15 tracking showed that 61.9% and 30% of the N-15, respectively, was removed from the system and assimilated by the macrophyte-biofilm complex after loading with 5.4 mg L-1 N-15 labelled NH4+ for 17 days. Nitrogen budget results showed that about 0.2%, 0.2% and 3.6% of the nitrogen were emitted as water-, HCl- and NaOH-soluble nitrogen-gas species, respectively. Bacteria (76.7-91.8%) were the predominant domain in all samples, followed by eukaryotes (8.0-23.0%), archaea and viruses. Network analyses showed that there were positive- and negative-correlative relationships among nitrogen-cycling genes and nitrifiers and denitrifiers. Our data highlight the important role of biofilm on submersed macrophytes for nitrogen removal. (C) 2020 Elsevier Ltd. All rights reserved.