Batch tests were carried out to analyze influences of the alkaline pretreatment and initial pH value on biohydrogen production from sewage sludge. Experimental results of the impact of different initial pH on biohydrogen production showed that both the maximal hydrogen yield occurred and that no methane was detected in the tests of at the initial pH of 11.0. The final pH decreased at the initial pH of 7.0-12.5 but increased atthe initial pH of 3.0-6.0, probably due to the combination of solubilized protein from sludge and the formation of volatile fatty acids (VFAs) and ammonia during biohydrogen fermentation. The performance of biohydrogen production using the raw sludge and the alkaline pretreated sludge was then compared in batch fermentation tests atthe initial pH of 11.0. The hydrogen yield was increased from 9.1 mL of H2/g of dry solids (DS) of the raw sludge to 16.6 mL of H2/g of DS of the alkaline pretreated sludge. No methane and less carbon dioxide (0.8% of control) were present in the biohydrogen production from the alkaline pretreated sludge. These results clearly showed that biohydrogen production could be enhanced and maintained stable by the combination of the high initial pH and alkaline pretreatment. The mechanism of biohydrogen production from sewage sludge at high initial pH was therefore investigated because the results of this study were differentfrom previous studies of biohydrogen production. Results showed that protein was the major substrate for biohydrogen production from sewage sludge and that Eubacterium multiforme and Paenibacillus polymyxa were the dominant bacteria in biohydrogen production from alkaline pretreated sludge at an initial pH of 11.0. The combination of alkaline pretreatment and high initial pH could not only maintain a suitable pH range for the growth of dominant hydrogen-producing anaerobes but also inhibit the growth of hydrogen-consuming anaerobes. In addition, the changes in pH value, oxidation-reduction potential, VFAs and soluble COD during hydrogen fermentation were also discussed.