A high-resolution record of Eocene paleo-temperature variation is preserved within the high southern latitude, marine shelf succession of the La Meseta Formation on Seymour Island, off the NE side of the Antarctic Peninsula. 87Sr/86Sr ratios of bivalve shell carbonate indicate that the La Meseta Formation spans virtually the entire Eocene, and suggest the presence of an early middle Eocene unconformity. Paleoclimatic and paleoceanographic inferences are based on the stable oxygen and carbon isotope values of two genera of bivalves collected with a high degree of stratigraphic resolution within the formation, and with multiple replicate samples from each horizon. δ18O data indicate roughly 10 °C of cooling from the early Eocene climatic optimum (~15 °C) through the end of the Eocene (minimum ~5 °C), much of which took place in two comparatively short intervals at ca. 52 and ca. 41 Ma. Many features of the isotope curves generated from this Eocene shelf section are apparent in δ18O and δ13C data from the Southern and global oceans, including warm intervals that likely correspond to the early and middle Eocene climatic optima (EECO and MECO). A rapid middle Eocene shift to much more positive values is the most significant in the section and reflects a drop to universally cooler temperatures in the late middle and late Eocene that might also be associated with a short-lived glacial advance. However, even using a somewhat depleted value for δ18O of seawater in the Antarctic peninsular region, average Seymour Island shelf-water paleotemperatures did not reach freezing before the end of the Eocene. δ13C data similarly reflect the documented middle Eocene surface ocean enrichment followed by more negative values, but depletion is much more pronounced on Seymour Island and persists for the remainder of the Eocene, suggesting a combination of upwelling, metabolic effects, and/or atypical carbon cycling on the shelf in this region. Isotope data capture information about changes in the paleoenvironment that also had consequences for the biota, as published paleontological records document marked change in the nature of terrestrial and marine biota at this time. The fact that middle Eocene cooling and biotic turnover in the Peninsular region correspond well in time to the proposed initial opening of Drake Passage suggests that the formation of gateways, in addition to changes in pCO2, had significant consequences for the Earth's climate system during the Paleogene.