Global cooling and the development of continental-scale Antarctic glaciation occurred in the late middle Eocene to early Oligocene (∼38 to 28 million years ago), accompanied by deep-ocean reorganization attributed to gradual Antarctic Circumpolar Current (ACC) development. Our benthic foraminiferal stable isotope comparisons show that a large δ13C offset developed between mid-depth (∼600 meters) and deep (>1000 meters) western North Atlantic waters in the early Oligocene, indicating the development of intermediate-depth δ13C and O2 minima closely linked in the modern ocean to northward incursion of Antarctic Intermediate Water. At the same time, the ocean's coldest waters became restricted to south of the ACC, probably forming a bottom-ocean layer, as in the modern ocean. We show that the modern four-layer ocean structure (surface, intermediate, deep, and bottom waters) developed during the early Oligocene as a consequence of the ACC.