The diffuse biomat formed on the bottom of shallow, open-water unit process wetland cells contains suboxic zones that provide conditions conducive to NO3(-) removal via microbial denitrification, as well as anaerobic ammonium oxidation (anammox). To assess these processes, nitrogen cycling was evaluated over a 3-year period in a pilot-scale wetland cell receiving nitrified municipal wastewater effluent. NO3(-) removal varied seasonally, with approximately two-thirds of the NO3(-) entering the cell removed on an annual basis. Microcosm studies indicated that NO3(-) removal was mainly attributable to denitrification within the diffuse biomat (i.e., 80 ± 20%), with accretion of assimilated nitrogen accounting for less than 3% of the NO3(-) removed. The importance of denitrification to NO3(-) removal was supported by the presence of denitrifying genes (nirS and nirK) within the biomat. While modest when compared to the presence of denitrifying genes, a higher abundance of the anammox-specific gene hydrazine synthase (hzs) at the biomat bottom than at the biomat surface, the simultaneous presence of NH4(+) and NO3(-) within the biomat, and NH4(+) removal coupled to NO2(-) and NO3(-) removal in microcosm studies, suggested that anammox may have been responsible for some NO3(-) removal, following reduction of NO3(-) to NO2(-) within the biomat. The annual temperature-corrected areal first-order NO3(-) removal rate (k20 = 59.4 ± 6.2 m yr(-1)) was higher than values reported for more than 75% of vegetated wetlands that treated water in which NO3(-) was the primary nitrogen species (e.g., nitrified secondary wastewater effluent and agricultural runoff). The inclusion of open-water cells, originally designed for the removal of trace organic contaminants and pathogens, in unit-process wetlands may enhance NO3(-) removal as compared to existing vegetated wetland systems.