This study evaluated the use of an anaerobic packed-bed reactor for hydrogen production at different hydraulic retention times (HRT) (1–8 h). Two reactors filled with expanded clay and fed with glucose (3136–3875 mg L−1) were operated at different total upflow velocities: 0.30 cm s−1(R030) and 0.60 cm s−1(R060). The effluent pH of the reactors was maintained between 4 and 5 by adding NaHCO3and HCl solutions. It was observed a maximum hydrogen production rate of 0.92 L H2 h−1 L−1in R030 at HRT of 1 h. Furthermore, the highest hydrogen yield of 2.39 mol H2 mol−1glucose was obtained in R060. No clear trend was observed by doubling the upflow velocities at this experiment. High ethanol production was also observed, indicating that the ethanol-pathway prevailed throughout the experiment.