Carbonates from approximately 2.3–2.1 billion years ago show markedly positive δ¹³C values commonly reaching and sometimes exceeding +10‰. Traditional interpretation of these positive δ¹³C values favors greatly enhanced organic carbon burial on a global scale, although other researchers have invoked widespread methanogenesis within the sediments. To resolve between these competing models and, more generally, among the mechanisms behind Earth’s most dramatic carbon isotope event, we obtained coupled stable isotope data for carbonate carbon and carbonate-associated sulfate (CAS). CAS from the Lomagundi interval shows a narrow range of δ³⁴S values and concentrations much like those of Phanerozoic and modern marine carbonate rocks. The δ³⁴S values are a close match to those of coeval sulfate evaporites and likely reflect seawater composition. These observations are inconsistent with the idea of diagenetic carbonate formation in the methanic zone. Toward the end of the carbon isotope excursion there is an increase in the δ³⁴S values of CAS. We propose that these trends in C and S isotope values track the isotopic evolution of seawater sulfate and reflect an increase in pyrite burial and a crash in the marine sulfate reservoir during ocean deoxygenation in the waning stages of the positive carbon isotope excursion.