This study assesses the impact of organic loading rate on biohydrogen production from glucose in an up-flow anaerobic packed bed reactor (UAnPBR). Two mesophilic UAPBRs (UAnPBR1 and 2) were tested at organic loading rates (OLRs) ranging from 6.5 to 51.4gCODL −1 d −1 . To overcome biomass washout, design modifications were made in the UAnPBR2 to include a settling zone to capture the detached biomass. The design modifications in UAnPBR2 increased the average hydrogen yield from 0.98 to 2.0mol-H 2 mol −1 -glucose at an OLR of 25.7gCODL −1 d −1 . Although, a maximum hydrogen production rate of 23.4±0.9LH 2 L −1 d −1 was achieved in the UAnPBR2 at an OLR of 51.4gCODL −1 d −1 , the hydrogen yield dropped by 50% to around 1mol-H 2 mol −1 -glucose. The microbiological analysis (PCR/DGGE) showed that the biohydrogen production was due to the presence of the hydrogen and volatile acid producers such as Clostridium beijerinckii, Clostridium butyricum, Megasphaera elsdenii and Propionispira arboris.