This paper investigated the improving mechanisms and microbial community dynamics of using zero-valent iron nanoparticles (Fe-0 NPs) in hydrogen fermentation of grass. Results showed that Fe-0 NPs supplement improved microbial activity and changed dominant microbial communities from Enterobacter sp. to Clostridium sp., which induced a more efficient metabolic pathway towards higher hydrogen production. Meanwhile, it is also proposed that Fe-0 NPs could accelerate electron transfer between ferredoxin and hydrogenase, and promote the activity of key enzymes by the released Fe2+. The maximal hydrogen yield and hydrogen production rate were 64.7 mL/g-dry grass and 12.1 mL/h, respectively at Fe-0 NPs dosage of 400mg/L, which were 73.1% and 128.3% higher compared with the control group. Fe-0 NPs also shorten the lag time and facilitated the hydrolysis and utilization of grass. This study demonstrated that Fe-0 NPs could effectively improve hydrogen production and accelerate the fermentation process of grass.