Life on Earth has been capitalizing on the C-3 photosynthetic pathway for 2.8 billion years. However, in the world's grasslands that emerged since the Paleogene, C-4 vegetation expanded dramatically between 8 and 3 Ma in response to climatic changes. Here we present the first comprehensive Late Miocene to Holocene delta C-13 soil carbonate record from the Eastern Mediterranean region (Anatolia) to reconstruct long-term geographic distributions of C-3 and C-4 plants, a region with patchy records compared to parts of Africa and Asia. Our results show a colonization of Anatolian floodplains by C-4 biomass by 9.9 Ma, similar to regions in NW and E Africa, followed by a transition from this mixed C-3-C-4 vegetation to C-4 dominance between ca. 7.1 and 4.9 Ma. The transition to C4 in Anatolia coincides with a similar shift from C-3 to C-4 vegetation in southern Asia and is generally attributed to the Late Miocene Cooling in response to decreasing atmospheric pCO(2). However, the Anatolian paleoecosystem patterns are unique due to a rapid and permanent return to C-3 dominance in the Early Pliocene, which is not observed elsewhere and occurs simultaneously with the disappearance of the open environment-adapted large mammal Pikermian chronofauna. We propose that this return to C-3 vegetation was caused by paleoclimatic processes that regionally shifted precipitation from the warm to the cool season, resembling the modern Mediterranean climate. In conclusion, changes in rainfall seasonality under subhumid climate, rather than increased aridity, drove the demise of C-4-dominated floodplains and the open-environment adapted Pikermian chronofauna at the Eurasian-African crossroads.