Several substrates were applied to evaluate the sulfate reducing bacteria(SRB) activity and sulfate removal from sulfate-rich mine drainage. Among the substrates, single cow manure and its mixture induced sulfate reduction; in addition, 90% of total sulfur was removed from mine drainage. The sulfates in Postgate medium added to these inocula were also reduced by 90% after 22 days. Although sulfide reduced by SRB existed in the form of H2S and HS？ in circumstantial pH, it was probably removed by gas dissociation of H2S because of low divalent metal concentrations in mine drainage. Inocula incubated onto Postgate media in anaerobic condition showed more heterotrophic anaerobe colony in mixture of cow manure and mushroom compost due to microbial diversity. The subsidiaries such as limestone and rice straw did not concerned SRB activity and removal of sulfur species, and it could be appled to substrate mixtures to enhance permeability and to prevent the channeling in full-scale passive treatment.