Paired benthic foraminiferal stable isotope and Mg/Ca data are used to estimate bottom water temperature (BWT) and ice volume changes associated with the Eocene-Oligocene Transition (EOT), the largest global climate event of the past 50 Myr. We utilized ODP Sites 1090 and 1265 in the South Atlantic to assess seawater δ18O (δw), Antarctic ice volume, and sea level changes across the EOT (∼33.8–33.54 Ma). We also use benthic δ13C data to reconstruct the sources of the deep water masses in this region during the EOT. Our data, together with previously published records, indicate that a pulse of Northern Component Water influenced the South Atlantic immediately prior to and following the EOT. Benthic δ18O records show a 0.5‰ increase at ∼33.8 Ma (EOT-1) that represents a ∼2°C cooling and a small (∼10 m) eustatic fall that is followed by a 1.0‰ increase associated with Oi-1. The expected cooling of deep waters at Oi-1 (∼33.54 Ma) is not apparent in our Mg/Ca records. We suggest the cooling is masked by coeval changes in the carbonate saturation state (Δ[CO32−]) which affect the Mg/Ca data. To account for this, the BWT, ice volume, and δw estimates are corrected for a change in the Δ[CO32−] of deep waters on the basis of recently published work. Corrected BWT at Sites 1090 and 1265 show a ∼1.5°C cooling coincident with Oi-1 and an average δw increase of ∼0.75‰. The increase in ice volume during Oi-1 resulted in a ∼70 m drop in global sea level and the development of an Antarctic ice sheet that was near modern size or slightly larger.