Earth's modern climate, characterized by polar ice sheets and large equator-to-pole temperature gradients, is rooted in environmental changes that promoted Antarctic glaciation similar to 33.7 million years ago. Onset of Antarctic glaciation reflects a critical tipping point for Earth's climate and provides a framework for investigating the role of atmospheric carbon dioxide (CO(2)) during major climatic change. Previously published records of alkenone-based CO(2) from high-and low-latitude ocean localities suggested that CO(2) increased during glaciation, in contradiction to theory. Here, we further investigate alkenone records and demonstrate that Antarctic and subantarctic data overestimate atmospheric CO(2) levels, biasing long-term trends. Our results show that CO(2) declined before and during Antarctic glaciation and support a substantial CO(2) decrease as the primary agent forcing Antarctic glaciation, consistent with model-derived CO(2) thresholds.