The feasibility and efficiency of magnetite nanoparticles (Fe(3)O(4)NPs) enhanced bio-hydrogen production from glucose anaerobic fermentation were evaluated in this study. The results demonstrated that the maximum hydrogen yield (HY) of 12.97 mL H-2/g-VSS was obtained with 50 mg/L and 40-60 nm of Fe(3)O(4)NPs in batch experiments. Moreover, the optimum dosage of Fe(3)O(4)NPs produced hydrogen production (HP) of 4.95 L H-2/d in an expanded granular sludge bed (EGSB) reactor. Fe(3)O(4)NPs involved could promote ethanol and acetic acid accumulation. Fe2+ as by-product of iron corrosion could effectively promote the activity of key coenzymes and soluble microbial products (SMPs). Importantly, Fe(3)O(4)NPs addition resulted in the formation of electronic conductor chains to enhance the electron transport efficiency in the granular sludge. Microbial community analysis revealed that the relative abundance of butyrate-hydrogen-producing bacteria (Clostridium) decreased from 40.55% to 11.45%, while the relative abundance of ethanol-hydrogen-producing bacteria (Acetanaerobacterium and Ethanoligenens) increased from 19.62% to 35.35% with Fe(3)O(4)NPs involved, confirming that the fermentation type was transformed from butyrate-type to ethanol-type, which finally facilitated more hydrogen production. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.