In this work, effect of Fe-0 nanoparticles (Fe-0 NPs) on macroalgae fermentation was explored. Hydrogen production was significantly enhanced by 6.5 times comparing with control test, achieving 20.25 mL H-2/g VSadded with addition of 200 mg/L Fe-0 NPs. In-depth analysis of substrate conversion showed that both hydrogen generation and acids accumulation were promoted with Fe-0 NPs supplementation. Microbial analysis demonstrated that both hydrogen-producing strains belonging to genus Clostridium and Terrisporobacter sp. favorable for acids formation were enriched with Fe-0 NPs supplementation, while species Acinetobacter lwoffii beneficial to organics mineralization was eliminated. Complex substrate compositions resulted in more prevalent cooperative relationships among species in the system. This study suggested that Fe-0 NPs plays a crucial role in macroalgae fermentation by affecting the microbial distribution, subsequently influencing the products distribution and energy conversion.