The enhancement of biohydrogen production from grass was investigated by addition of Fe2+, and the microbial community, microbial activity and kinetic models analysis were used to explore the improving mechanisms of Fe2+ addition. The results showed that Fe2+ could significantly improve hydrogen fermentation efficiency of grass. The highest hydrogen yield achieved 72.8 mL/g-dry grass at the Fe2+ addition of 400 mg/L, which was 49.6% higher in comparison with the control group (48.7 mL/g-dry grass). Organics utilization was also improved from 15.9% to 20.6% at the Fe2+ addition of 400 mg/L. Fe2+ changed the metabolic pathway towards more efficient hydrogen production and accelerated the hydrolysis of grass, thereby enhancing the conversion process of substrates to hydrogen. Furthermore, microbiological analysis showed that Fe2+ improved the microbial activity and enriched more hydrogen-producers, and changed the dominant H-2-producer from Enterobacter to Clostridium. The addition of Fe2+ also significantly decreased the presence of H-2-competitor Enterococcus. This study demonstrated that the addition of Fe2+ was an effective and a simple strategy to improve fermentative hydrogen production from grass.