This study evaluated the hydrogen and methane production from sugarcane vinasse in an anaerobic fluidized bed reactor. Two reactors were operated with two different substrate concentrations: R5 (5 g COD L-1) and R10 (10 g COD L-1). During the first stage, glucose was used as the primary carbon source; vinasse was then added from 0% to 100% of the organic source in hydraulic retention time (HRT) of 6 h. Later, HRT was changed to 4, 2 and 1 h. The best hydrogen production rate was 0.57 L h(-1) L-1 (R5, HRT = 1 h, 100% vinasse). The best hydrogen yield was 3.07 mmol H-2 g(-1) CODadded (R5, HRT = 6h, vinasse:glucose = 1:3). Main metabolites were ethanol, butyric acid, propionic acid and methanol. Denaturing gradient gel electrophoresis analysis identified Prevotella sp. and Megasphaera sp. belonging to the Bacteria domain and Methanobacterium sp. and Methanosphaera sp. belonging to the Archaea domain. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.