Heterotrophic denitrifying enriched culture (DEC) from a lab-scale high-rate denitrifying reactor was discovered to perform nitrate-dependent anaerobic ferrous oxidation (NAFO). The DEC was systematically investigated to reveal their denitrification activity, their NAFO activity, and the predominant microbial population. The DEC was capable of heterotrophic denitrification with methanol as the electron donor, and autotrophic denitrification with ferrous salt as the electron donor named NAFO. The conversion ratios of ferrous-Fe and nitrate-N were 87.41 and 98.74 %, and the consumption Fe/N ratio was 2.3:1 (mol/mol). The maximum reaction velocity and half saturation constant of Fe were 412.54 mg/(l h) and 8,276.44 mg/l, and the counterparts of N were 20.87 mg/(l h) and 322.58 mg/l, respectively. The predominant bacteria were Hyphomicrobium, Thauera, and Flavobacterium, and the predominant archaea were Methanomethylovorans, Methanohalophilus, and Methanolobus. The discovery of NAFO by heterotrophic DEC is significant for the development of wastewater treatment and the biogeochemical iron cycle and nitrogen cycle.