Effects of biochar (BC) and ferrous iron (Fe2+) additions on hydrogen (H 2 ) production from glucose were investigated using batch experiment. The glucose with both BC and Fe2+ additions were incubated at 37 °C for H 2 production. As compared with the control group (without BC and Fe2+ additions), the synergic effects of BC and Fe2+ make the lag phase time decease from 4.25 to 2.12 h, and H 2 yield increase from 158.0 to 234.4 ml/g glucose. Moreover, suitable concentrations of BC and Fe2+ serve to enhance volatile fatty acid generation during H 2 evolution. These results indicate that H 2 production is improved by BC and Fe2+ regulations, where synergic mechanisms are described as follows: BC acts as support carriers of anaerobes and system pH buffers, which promotes the biofilm formation and maintains suitable pH environment; Appropriate Fe2+ concentration can improve hydrogenase activity in H 2 production.