We demonstrate that the carbon isotopic signal of mid-depth waters evolved differently from deep waters in the South Atlantic sector of the Southern Ocean during the Pleistocene. Deep sites (>3700 m) exhibit large glacial-to-interglacial variations in benthic δ13C, whereas the amplitude of the δ13C signal at Site 1088 (∼2100 m water depth) is small. Unlike the deep sites, at no time during the Pleistocene were benthic δ13C values at Site 1088 lower than those of the deep Pacific. Reconstruction of intermediate-to-deep δ13C gradients (Δ13CI-D) supports the existence of a sharp chemocline between 2100 and 2700 m during most glacial stages of the last 1.1 myr. This chemical divide in the glacial Southern Ocean separated well-ventilated water above ∼2500 m from poorly ventilated water below. The Δ13CI-D signal parallels the Vostok atmospheric pCO2 record for the last 400 kyr, lending support to physical models that invoke changes in Southern Ocean deep water ventilation as a mechanism for changing atmospheric pCO2. The emergence of a strong 100-kyr cycle in Δ13CI-D during the mid-Pleistocene supports a change in vertical fractionation and deep-water ventilation rates in the Southern Ocean, and is consistent with possible CO2-forcing of this climate transition.