The marine foraminiferal isotope record displays a positive δ18O shift early in the Oligocene, which has been identified as the onset of the Antarctic Oi-1 glaciation. Reported here are the first oxygen isotope–derived summer paleotemperatures for continental freshwater in the Northern Hemisphere (Hampshire Basin, Isle of Wight, UK) leading up to and across this event. These paleotemperatures are derived from multiple paleoproxies (rodent tooth enamel, gastropod shells, charophyte gyrogonites, and fish otoliths) and are independent of freshwater evaporation effects and changes in ice volume. We conclude that a fluctuating mesothermal climate existed, but that there was no significant decrease in summer temperatures across the Oi-1 glaciation. This result is concordant with several other studies in suggesting that the majority of the isotopic shift in the marine realm across the Oi-1 glaciation is linked to changes in Antarctic ice volume and not to global temperature change. Our new approach has allowed us to derive numerical values for summer temperatures as well as to reconstruct relative temperature change across this key interval of the Eocene-Oligocene transition.