The Eocene-Oligocene transition (EOT; ca. 33–34 Ma) was a time of pronounced climatic change, marked by the establishment of continental-scale Antarctic ice sheets. The timing and extent of temperature change associated with the EOT is controversial. Here we present multiproxy EOT climate records (∼15–34 k.y. resolution) from St. Stephens Quarry, Alabama, USA, derived from foraminiferal Mg/Ca, δ18O, and TEX86. We constrain sea-surface temperatures (SSTs) in the latest Eocene and early Oligocene and address the issue of climatic cooling during the EOT. Paleotemperatures derived from planktic foraminifera Mg/Ca and TEX86 are remarkably consistent and indicate late Eocene subtropical SSTs of >28 °C. There was substantial and accelerated cooling of SSTs (3–4 °C) through the latest Eocene “precursor” δ18O shift (EOT-1), prior to Oligocene Isotope-1 (Oi-1). Our multispecies planktic foraminiferal δ18O records diverge at the E/O boundary (33.7 Ma), signifying enhanced seasonality in the earliest Oligocene in the Gulf of Mexico.