Start-up of a continuously stirred tank reactor for bio-hydrogen production under different initial organic loading rate (OLR) of 3, 7 and 10 kgCOD/m3 d, respectively, was carried out with sewage sludge as inoculum. Molasses wastewater was used as substrate and hydraulic retention time was kept at 6 h. This study aimed to assess OLR on the formation of fermentation types and the structure of microbial communities during the start-up period. It was found that at an initial OLR of 7 kgCOD/m3 d and an initial biomass of 6.24 gVSS/L, an equilibrial microbial community of ethanol-type fermentation could be established within 30 days. The observed average specific hydrogen production rate was 276 mLH2/gVSS d, which was 40% higher than that of the one acclimated with 3 kgCOD/m3 d. Based on denaturing gradient gel electrophoresis profiles, significant microbial population shifts took place at the first 15 days, but a longer period up to 30 days was required to establish a microbial community with stable metabolic activity.