A detailed record of the early-Earth sulfur (S) cycle is chronicled by the S isotope values of sulfide and sulfate preserved in the rock record. Interpretation of this record rests on our understanding of sulfur cycling in modern systems, experiments, and the resulting isotopic signatures. Very large fractionations in δ34S of ≥70‰ are commonly measured between sulfide and sulfate in modern systems and in ancient sediments. Theoretical calculations suggest that sulfate-reducing prokaryotes are capable of producing such large fractionations during the reduction of sulfate to sulfide, although they have only been demonstrated to generate fractionations up to 48‰. Here we report the first direct determination of 60‰–70‰ fractionations by natural populations of sulfate reducers. These high fractionations occur under the relatively low-sulfate conditions (1.1–2 mM) of meromictic Lago di Cadagno in Switzerland. The major and minor isotopic composition of sulfide and sulfate in the lake water is consistent with sulfide produced by sulfate reduction, with little evidence for modification by further oxidative sulfur cycling. These observations help us to constrain the evolution of seawater sulfate concentrations.