Conductive nano-materials have been reported to accelerate methanogenesis by promoting direct interspecies electron transfer (DIET), while their effects seem to vary depending on operational conditions. The present study examined effects of magnetite nanoparticles (MNPs) on methanogenesis from acetate by soil-derived anaerobic cultures under continuous agitation. We found that MNPs accelerated methanogenesis in agitated cultures as has been observed previously for static cultures. Metabarcoding of 16S rRNA gene amplicons showed that substantially increased in the presence of MNPs, while DIET-related did not occur. Metagenomic and metatranscriptomic analyses confirmed the predominance of in MNP-supplemented agitated cultures. In addition, genes coding for acetoclastic methanogenesis but not those for hydrogenotrophic methanogenesis were abundantly expressed in the dominant in the presence of MNPs. These results suggest that MNPs stimulate acetoclastic methanogenesis under continuous agitation. Previous studies have shown that conductive nano particles, such as MNPs, accelerate methanogenesis and suggested that MNPs facilitate DIET between exoelectrogenic bacteria and methanogenic archaea. In these methanogens, electron thus obtained are considered to be used for hydrogenotrophic methanogenesis. However, the present work provides evidence that shows that MNPs accelerate DIET-independent acetoclastic methanogenesis under continuous agitation. Since most of previous studies have examined effects of MNPs in static or weakly agitated methanogenic cultures, results obtained in the present work suggest that hydraulic conditions definitively determine how MNPs accelerate methanogenesis. Besides, the knowledge obtained in this study is useful for engineers operating stirred-tank anaerobic digesters, since we show that MNPs accelerate methanogenesis under continuous agitation.