The Eocene-Oligocene transition towards a cool climate (∼33.5 million years ago) was one of the most pronounced climate events during the Cenozoic era1. The marine record of this transition has been extensively studied. However, significantly less research has focused on continental climate change at the time, yielding partly inconsistent results on the magnitude and timing of the changes2-8. Here we use a combination ofin vivostable isotope compositions of fossil tooth enamel with diagenetic stable isotope compositions of fossil bone to derive a high-resolution (about 40,000 years) continental temperature record for the Eocene-Oligocene transition. We find a large drop in mean annual temperature of 8.2 ± 3.1 °C over about 400,000 years, the possibility of a small increase in temperature seasonality, and no resolvable change in aridity across the transition. The large change in mean annual temperature, exceeding changes in sea surface temperatures at comparable latitudes9,10and possibly delayed in time with respect to marine changes by up to 400,000 years, explains the faunal turnover for gastropods, amphibians and reptiles, whereas most mammals in the region were unaffected. Our results are in agreement with modelling studies that attribute the climate cooling at the Eocene-Oligocene transition to a significant drop in atmospheric carbon dioxide concentrations.