The perennial springs at Gypsum Hill (GH) and Colour Peak (CP), situated at nearly 80°N on Axel Heiberg Island in the Canadian high Arctic, are one of the few known examples of cold springs in thick permafrost on Earth. The springs emanate from deep saline aquifers and discharge cold anoxic brines rich in both sulfide and sulfate. Grey-coloured microbial streamers form during the winter months in snow-covered regions of the GH spring run-off channels (−1.3°C to 6.9°C, ∼7.5% NaCl, 0–20 p.p.m. dissolved sulfide, 1 p.p.m. dissolved oxygen) but disappear during the Arctic summer. Culture- and molecular-based analyses of the 16S rRNA gene (FISH, DGGE and clone libraries) indicated that the streamers were uniquely dominated by chemolithoautotrophic sulfur-oxidizing Thiomicrospira species. The streamers oxidized both sulfide and thiosulfate and fixed CO2 under in situ conditions and a Thiomicrospira strain isolated from the streamers also actively oxidized sulfide and thiosulfate and fixed CO2 under cold, saline conditions. Overall, the snow-covered spring channels appear to represent a unique polar saline microhabitat that protects and allows Thiomicrospira streamers to form and flourish via chemolithoautrophic, phototrophic-independent metabolism in a high Arctic winter environment characterized by air temperatures commonly below −40°C and with an annual average air temperature of −15°C. These results broaden our knowledge of the physical and chemical boundaries that define life on Earth and have astrobiological implications for the possibility of life existing under similar Martian conditions.