In this work, the effects of granular activated carbon (GAC) supplementation on hydrogen fermentation were investigated in Clostridium butyricum. Maximum hydrogen production rate and ultimate hydrogen volume increased up to 1.7 and 1.9 fold, respectively, with 1 g/L GAC supplementation. Indicators of stable hydrogen production, such as acetate and butyrate formation increased with increasing GAC concentration. To identify the factors for hydrogen production enhancement, transcriptome analysis was conducted. Functional genes related to hydrogen production increased by GAC supplementation (1.75 fold for pfor and 1.48 fold for oxct). On the other hand, functional genes related to hydrogen consumption decreased (1.78 fold for ldh, 0.67 fold for adh, 1.64 fold for hbd, 2.4 fold for crt, and 0.71 fold for buk). Considered together, these results suggested that GAC supplementation increased biohydrogen production by changing the metabolic flux associated with hydrogen production and consumption pathways.