Biohydrogen production using the high-rate reactor is promising due to its ability of maintaining higher biomass concentrations through forming granules or biofilms. This study investigated the effect of reaction mode on hydrogen fermentation by comparing an upflow anaerobic sludge blanket (UASB) and a packed bed reactor (PBR). UASB and PBR were operated for 120 days at hydraulic retention time (HRT) of 24-12 h and organic loading rates (OLRs) of 0.96-15.36 g COD/Lid. Both UASB and PBR achieved maximal hydrogen production rates as 2.77 +/- 0.18 and 1.28 +/- 0.12 L/L/d, respectively, at 15.36 g COD/Lid, corresponding to hydrogen yields of 1.44 +/- 0.01 and 0.67 +/- 0.06 mol/mol glucose. Illumina MiSeq sequencing results revealed Clostridium sp. was the dominant microbial consortium for hydrogen production in UASB (92.1%) and PBR (71.7%). Compared to UASB, PBR showed a greater microbial diversity of ethanol and lactic acid producers, and may be more favorable for methanogenesis and homoacetogenesis. This study demonstrates that reaction mode significantly influenced microbial diversity and biohydrogen production. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.