The nitrate-dependent anaerobic ferrous oxidizing (NAFO) is a newly discovered bioprocess, which is valuable to develop autotrophic denitrifying technology. In this work, the physicochemical characteristics and microbial community of the cultivated sludge (NAFO sludge) from a lab-scale reactor operating for over 200 days were investigated. A comparison between the seeding sludge and NAFO sludge was also carried out. The results showed that the NAFO sludge possessed distinctly high settleability, and the average settling velocity was 390.1 +/- 161.3 m/h. The increasing particle size and density, with average values of 1469 +/- 65 mu m (surface area-weighed mean diameter, SMD)/2389 +/- 132 mu m (volumetric-weighed mean diameter, VMD) and 1580 +/- 80 kg/m(3), were revealed to the main factors causing the profile of excellent settleability. High content of iron with peak value of 87.5% (wt) was detected in NAFO sludge, which was mainly in the form of oxide compounds, such as hematite and magnetite, endowing unique morphology characteristics of NAFO sludge. After the long-term operation, a microbial succession was supposed to occur in the granular sludge, and the dominant bacterial communities in the NAFO sludge were Gamma-proteobacteria, Thermoleophilia, Clostridia and Alpha-proteobacteria classes. The present work will benefit the development of NAFO technology, and also the separation and iron-recycling processes of the NAFO sludge. (C) 2016 Elsevier B.V. All rights reserved.