© 2014, American Association for the Advancement of Science. All rights reserved. The minor extent of sulfur isotope fractionation preserved in many Neoarchean sedimentary successions suggests that sulfate-reducing microorganisms played an insignificant role in ancient marine environments, despite evidence that these organisms evolved much earlier. We present bulk, microdrilled, and ion probe sulfur isotope data from carbonate-associated pyrite in the ∼2.5-billion-year-old Batatal Formation of Brazil, revealing large mass-dependent fractionations (approaching 50 per mil) associated with microbial sulfate reduction, as well as consistently negative Δ 33S values (∼ -2 per mil) indicative of atmospheric photochemical reactions. Persistent 33S depletion through ∼60 meters of shallow marine carbonate implies long-term stability of seawater sulfate abundance and isotope composition. In contrast, a negative Δ 33S excursion in lower Batatal strata indicates a response time of ∼40,000 to 150,000 years, suggesting Neoarchean sulfate concentrations between ∼1 and 10 μM.