The biogeochemical sulfur cycle plays a central role in fueling microbial metabolisms, regulating the Earth’s redox state, and affecting climate. However, geochemical reconstructions of the ancient sulfur cycle are confounded by ambiguous isotopic signals. We use phylogenetic reconciliation to ascertain the timing of ancient sulfur cycling gene events across the tree of life. Our results suggest that metabolisms using sulfide oxidation emerged in the Archean, but those involving thiosulfate emerged only after the Great Oxidation Event. Our data reveal that observed geochemical signatures resulted not from the expansion of a single type of organism but were instead associated with genomic innovation across the biosphere. Moreover, our results provide the first indication of organic sulfur cycling from the Mid-Proterozoic onwards, with implications for climate regulation and atmospheric biosignatures. Overall, our results provide insights into how the biological sulfur cycle evolved in tandem with the redox state of the early Earth.