Sulphate-reducing microbes affect the modern sulphur cycle, and may be quite ancient, though when they evolved is uncertain. These organisms produce sulphide while oxidizing organic matter or hydrogen with sulphate. At sulphate concentrations greater than 1 mM, the sulphides are isotopically fractionated (depleted in 34S) by 10-40/1000 compared to the sulphate, with fractionations decreasing to near 0/1000 at lower concentrations. The isotope record of sedimentary sulphides shows large fractionations relative to seawater sulphate by 2.7 Gyr ago, indicating microbial sulphate reduction. In older rocks, however, much smaller fractionations are of equivocal origin, possibly biogenic but also possibly volcanogenic. Here we report microscopic sulphides in approximately 3.47-Gyr-old barites from North Pole, Australia, with maximum fractionations of 21.1/1000, about a mean of 11.6/1000, clearly indicating microbial sulphate reduction. Our results extend the geological record of microbial sulphate reduction back more than 750 million years, and represent direct evidence of an early specific metabolic pathway--allowing time calibration of a deep node on the tree of life.