Bio-hydrogen production from sugarcane vinasse in anaerobic up-flow packed-bed reactors (APBR) was evaluated. Four types of support materials, expanded clay (EC), charcoal (Ch), porous ceramic (PC), and low-density polyethylene (LDP) were tested as support for biomass attachment. APBR (working volume - 2.3 L) were operated in parallel at a hydraulic retention time of 24 h, an organic loading rate of 36.2 kg-COD m(-3) d(-1), at 25 °C. Maximum volumetric hydrogen production values of 509.5, 404, 81.4 and 10.3 mL-H2 d(-1) L(-1)reactor and maximum yields of 3.2, 2.6, 0.4 and 0.05 mol-H2 mol(-1) carbohydrates total, were observed during the monitoring of the reactors filled with LDP, EC, Ch and PC, respectively. Thus, indicating the strong influence of the support material on H2 production. LDP was the most appropriate material for hydrogen production among the materials evaluated. 16S rRNA gene by Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis and scanning electron microscopy confirmed the selection of different microbial populations. 454-pyrosequencing performed on samples from APBR filled with LDP revealed the presence of hydrogen-producing organisms (Clostridium and Pectinatus), lactic acid bacteria and non-fermentative organisms.