We examined nitrate-dependent Fe2+ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of "Candidatus Brocadia sinica" anaerobically oxidized Fe2+ and reduced NO3- to nitrogen gasat rates of 3.7±0.2 and 1.3±0.1 (mean±standard deviation [SD]) nmol mg protein-1 min-1, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of "Ca. Brocadia sinica" (10 to 75 nmol NH4+ mg protein-1 min-1). A 15N tracer experiment demonstrated that coupling of nitrate-dependent Fe2+ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO3- by "Ca. Brocadia sinica." The activities of nitrate-dependent Fe2+ oxidation were dependent on temperature and pH, and thehighest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO3 -±SD of "Ca. Brocadia sinica" was determined to be 51±21 μM. Nitrate-dependent Fe2+ oxidation was further demonstrated by another anammox bacterium, "Candidatus Scalindua sp.," whose rates of Fe2+ oxidation and NO3- reduction were 4.7±0.59 and 1.45±0.05 nmol mg protein-1 min-1, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe2+ oxidation and the anammox reaction decreased the molar ratios of consumed NO2 - to consumed NH4 + (δNO2-/δNH4 +) and produced NO3- to consumed NH4 + (δNO3 -/δNH4 +). These reactions are preferable to the application of anammox processes for wastewater treatment.