Abstract-This study examines fermentative hydrogen production from untreated (raw) sludge and pretreated sludge by sterilization treatment (15, 30, 45, and 60 min) as substrate using mixed cultures in batch experiments under anaerobic thermophilic conditions. Longer treatment time was found to be highly effective for hydrolyzing organic matters in the sludge. Soluble chemical oxygen demand (SCOD) of pretreated sludge was 1.2 to 1.9-fold higher than that of untreated sludge. Sterilization treatment was found to accelerate and increase hydrogen production throughout the batch mode, but with no measurable methane production. Pretreated (30 min) sludge presented an optimal condition, resulting in maximum hydrogen yield (25.1 ml H 2 /g-VS) and the highest hydrogen content (60.0%). Under the same conditions, enhanced hydrogen yield was 6.4-fold higher, which came with an additional benefit of efficient VS removal over the use of untreated sludge. This was attributed to destruction of solids in sludge during the solubilization process. Present findings have potential practical use in not only processes for efficient hydrogen production via anaerobic fermentation but also in waste treatment.