The existence of oxidative microbial sulfur cycling in the deep biosphere has been postulated, but it is difficult to identify because isotope effects of reductive sulfur cycling generally overprint those of oxidative sulfur cycling. In the sediments at Integrated Ocean Drilling Program (IODP) Site C0007 (Holes A–C) drilled and sampled during IODP Expedition 316 at the Nankai Trough, Japan, sulfur and oxygen isotope compositions of dissolved and solid-phase sulfur compounds show evidence for a discrete zone of deep oxidative sulfur cycling. The sulfate concentration profile and isotope values show two distinct sulfate reduction regimes: one for downward-diffusing sulfate in the uppermost 36 m of sediment (unit I), and another zone below 90 m, where sulfate diffuses upward from a deep subsurface fluid. The sulfur and oxygen isotope composition of sulfate and a dramatic change in solid-phase iron geochemistry indicate that oxidative sulfur cycling occurs at depth. The highly dynamic sedimentary and tectonic regime at Nankai Trough has created an environment in the subsurface where geochemical gradients can sustain deep oxidative sulfur cycling over long durations of time.