The present study evaluated the influence of upflow velocity (V-up) applied to an anaerobic Fluidized-bed reactor used for hydrogen production. For comparison, two reactors were used with different velocities: 1.24 cm s(-1) (R124) and 1.88 cm s(-1) (R188). Expanded clay was used as a support material for immobilization along with synthetic wastewater containing glucose as the main carbon source (5000 mg L-1). The reactor R124, which operated at the minimum fluidization velocity (1.24 cm s(-1)), had the best values for hydrogen production. The maximum hydrogen production rate was 2.21 L h(-1) L-1 for a hydraulic retention time (HRT) of 1 h, while the best hydrogen yield was 2.55 mol H-2 mol(-1) glucose for an HRT of 2 h. The hydrogen content in the biogas was around 40.53-67.57%. A high amount of ethanol was produced, suggesting that a metabolic pathway via ethanol was preferable. In general, the reactor under the minimum velocity fluidization (R124) presented a greater production of hydrogen than R188 showing that should have a limit point in Vup till the hydrogen production is maximized. (C) 2011 Elsevier B.V. All rights reserved.