The Paint Pots are a natural Fe–SO4 acid spring system along the Kicking Horse Rim, a major geological feature that has controlled fluid flow and mineralization over geologic time. The very low pH (∼3) and extremely high trace metal concentrations of the springs are anomalous and greatly exceed health limits (Zn = 35.8 mg/L, Pb = 0.461 mg/L, As = 82.7 ppb). Sulphur isotopes (δ34S and δ18O in sulphate equal to +11.4 and –12.1 ‰, respectively) are consistent with sulphide oxidation. Mass balance calculations indicate that the springs must have derived their acidity and metal content from natural weathering of a pyrite-rich ore deposit in the vicinity. Several ore deposit types occur along the Kicking Horse Rim, with the Mississippi Valley Type (MVT) style being the most consistent with the source of the spring waters. At the spring outlet, Fe-rich waters oxidize, forming large cone features of Fe–O precipitates, predominantly goethite. A diverse microbial community has been identified in the site that is distinct from those found at acid mine drainage sites. At one site, there was a high proportion of the bacterial candidate division WPS-2, the first time that this group has been detected as the predominant phylum in a community. Compared with anthropogenically influenced acid mine drainage sites that have been studied to date as Mars analogues, the unique uncultured organisms found in the Paint Pots provide a distinct analogue site that can offer insights into the diversity of extremophilic organisms in more natural environments.