The Eocene-Oligocene (E-O) climate transition (ca. 34 Ma) marks a period of Antarctic ice growth and a major step from early Cenozoic greenhouse conditions toward today's glaciated climate state. The transition is represented by an increase in deep-sea benthic foraminiferal oxygen isotope (δ18O) values occurring in two main steps that reflect the temperature and δ18O of seawater. Existing benthic Mg/Ca paleotemperature records do not display a cooling across the transition, possibly reflecting a saturation state effect on benthic foraminiferal Mg/Ca ratios at deep-water sites. Here we present data from exceptionally well preserved foraminifera deposited well above the calcite compensation depth that provide the first proxy evidence for an ∼2.5 °C ocean cooling associated with the ice growth. This permits interpretation of E-O δ18O records without invoking Northern Hemisphere continental-scale ice.