The acidophilic heterotrophic bacteria Acidiphilium spp. were considered as a ministrant of Leptospirillum spp., Acidithiobacillus spp. and other autotrophic bacteria in acid mine drainage (AMD). 25 Acidiphilium-like bacterial strains were isolated from eight different mines in China and their physiology was characterized. Their marmatite-bioleaching ability has been investigated and compared with that of a mixed culture of Acidiphilium sp. and Acidithiobacillus ferrooxidans. The results indicated that some Acidiphilium-like strains exhibited a high marmatite-bioleaching ability, which was even higher than that of Acidithiobacillus ferrooxidans. The leaching efficiency was not higher in mixed than in pure culture. The phylogenetic relationship of the 25 Acidiphilium-like strains was analyzed by 16S rRNA sequencing, GyrB sequencing and rep-PCR genomic fingerprinting. The 25 strains and other eight species of Acidiphilium spp. were clustered into three groups.