The relation between the partial pressure of atmospheric carbon dioxide (pCO₂) and Paleogene climate is poorly resolved. We used stable carbon isotopic values of di-unsaturated alkenones extracted from deep sea cores to reconstruct pCO₂ fromthe middle Eocene to the late Oligocene (∼45 to 25 million years ago). Our results demonstrate that pCO₂ ranged between 1000 to 1500 parts per million by volume in the middle to late Eocene, then decreased in several steps during the Oligocene, and reached modern levels by the latest Oligocene. The fall in pCO₂ likely allowed for a critical expansion of ice sheets on Antarctica and promoted conditions that forced the onset of terrestrial C₄ photosynthesis.