Nitrate-dependent anaerobic iron oxidation (NAIO) is a chemolithotrophic bioprocess that converts nitrate to nitrogen gas using zero-valent/ferrous iron, making it valuable for autotrophic denitrification. Seeding sludge is vital for the application of NAIO process, but scarce information is available about the capability of active sludge. In this work, activated sludge (AS) and anaerobic granular sludge (AGS) were investigated for their NAIO performance, and AGS was preferred as inocula after systematic evaluation on three aspects. AGS demonstrated higher nitrogen removal performance with nitrate removal rate of 0.20 +/- 0.02 kg-N/(m(3).d) and removal efficiency of 95.58 +/- 2.29%. The AGS system showed robustness facing NAIO operation conditions, exhibiting stable sludge characteristics (e.g. particle size and shape factors) with variation range of -14.75% to +26.43%. The AGS system demonstrated high economical efficiency with lower iron consumption for nitrate removal and higher iron content for resource recycling. 16S rRNA gene high-throughput sequencing was applied to investigate the microbial community. Nitrosomonadales_unclassified, Geothrix in AS and Nitrosomonadales_unclassified, Desulforhabdus in AGS were supposed to play important roles in NAIO process. The present work clarifies about the choice of seeding sludge for the application of the chemolithotrophic NAIO process and for the development of biotechnology.