A major obstacle in understanding the evolution of Cenozoic climate has been the lack of well dated terrestrial evidence from high-latitude, glaciated regions. Here, we report the discovery of exceptionally well preserved fossils of lacustrine and terrestrial organisms from the McMurdo Dry Valleys sector of the Transantarctic Mountains for which we have established a precise radiometric chronology. The fossils, which include diatoms, palynomorphs, mosses, ostracodes, and insects, represent the last vestige of a tundra community that inhabited the mountains before stepped cooling that first brought a full polar climate to Antarctica. Paleoecological analyses, (40)Ar/(39)Ar analyses of associated ash fall, and climate inferences from glaciological modeling together suggest that mean summer temperatures in the region cooled by at least 8 degrees C between 14.07 +/- 0.05 Ma and 13.85 +/- 0.03 Ma. These results provide novel constraints for the timing and amplitude of middle-Miocene cooling in Antarctica and reveal the ecological legacy of this global climate transition.