Zero-valent iron (ZVI) supplementation and two-stage anaerobic digestion are the enhanced ways for CH4 production in anaerobic treatment to convert organic wastes into renewable energy. In this study, different ZVI supplementation modes were applied during the acidogenic stage or methanogenic stage in swine manure anaerobic digestion. Adding ZVI in the acidogenic stage can increase the pH and improve volatile fatty acid production by 16.5%; ZVI addition in the methanogenic stage is more beneficial for CH4 production than in the acidogenic stage. The results show that adding ZVI at both the acidogenic stage and methanogenic stage could achieve 27.3% CH4 yield higher than that of control. The microbial sequencing analysis showed that ZVI supplementation could enrich hydrogenotrophic Methanomassiliicoccus by 39.2-92.0%. Results from this study demonstrated that ZVI could be added to contact directly with methanogens in order to achieve higher energy recovery from animal manure.