We experimentally and theoretically investigated implications of H-2 and a rate-limiting step in a mixed-culture biofilm anode fed with n-butyrate, one of the poorest substrates to exoelectrogens. Acetate and i-butyrate were formed as intermediates during anaerobic degradation of n-butyrate, which suggested oxidative acetogenesis of n-butyrate in syntrophy with H-2 scavengers in the biofilm anode. Methane was not detected in an anode chamber, and no current was generated in the biofilm anode using H-2 as the electron donor. These results indicated that acetogens would be a main H-2 consumer in the biofilm. Pyrosequencing data showed dominance of Geobacter in the biofilm anode (83.6% of total sequences), along with Sphaerochaeta and Treponema, which supports the syntrophy between exoelectrogens and acetogens. Electrical conductivity of the butyrate-fed biofilm anode was as high as 0.67 mS/cm, demonstrating that EET does not limit current density in the biofilm. In-situ monitoring of dissolved H-2 concentration proved H-2 production (up to 12.4 mu M) and consumption during current generation in the biofilm, which supports significance of H-2-based syntrophy in the electrically conductive biofilm using n-butyrate as the primary electron donor.