The effects of pretreatment method of cow dung compost, which was employed as natural hydrogen bacteria source, on the microbial community, population distribution of microbes and hydrogen production potential were investigated in the batch tests. The maximum hydrogen yield of 290.8 mL/L-culture appeared in the pretreated method A (infrared drying) by dark fermentation. The pretreated method of compost significantly affected microbial succession, population distribution of microbes. Both Clostridium sp. and Enterobacter sp. were found to be two species of preponderant hydrogen-producing bacteria, the next best was Bacteroides sp. and Veillonella sp., the last was Lactobacillus sp. and Streptococcus sp., which were also essential. The results showed that the mutualism and symbiosis relations of the mixed bacteria played a critical role in hydrogen fermentation process. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.