The Grande Coupure corresponds to a major episode of faunal turnover in western Europe around the Eocene-Oligocene boundary that is generally attributed to the influx of multiple clades of Asian mammals. However, Asian mammal clades begin to appear in the fossil record of southeastern Europe during the middle Eocene, 5–10 million years prior to the Grande Coupure. How and when these Asian mammal clades colonized southeastern Europe remains poorly understood, partly because the fossil record of mammals from nearby Anatolia is characterized by marked endemism and very limited exchanges with Asia during most of the Eocene. We resolve this apparent paradox by reviewing the age of existing paleontological sites from the Balkans to the Caucasus and documenting the oldest Asian perissodactyls found so far in central Anatolia, which date to the lower or middle Priabonian, 37.8 to 35 million years ago, on the basis of geochronological, magnetostratigraphic and biostratigraphic data. We show that the Eocene distribution of mammals across Eurasia supports a previously unrecognized biogeographic province, designated here as Balkanatolia, spanning the eastern and central segment of the Neotethyan margin. Isolated from mainland Eurasia during the early and middle Eocene, Balkanatolia formed a low-topography archipelago where endemic and anachronistic mammals thrived. We show that the Eocene fossil record supports Balkanatolia having been colonized by Asian ungulates and rodents by the late Bartonian (mammalian Paleogene biohorizon MP16), following the establishment of a continuous terrestrial dispersal corridor across the central segment of the Neotethyan margin. This colonization event was facilitated by a drop in global eustatic sea level and a tectonically-driven sea retreat in eastern Anatolia and the Lesser Caucasus during the late middle Eocene. These paleogeographic changes instigated the demise of Balkanatolia as a distinct biogeographic province and paved the way for the dispersal of Asian endemic clades before and during the Grande Coupure in western Europe.