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 ~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 (CO2) during major climatic change. Previously published records of alkenone-based CO2 from high- and low-latitude ocean localities suggested that CO2 increased during glaciation, in contradiction to theory. Here, we further investigate alkenone records and demonstrate that Antarctic and subantarctic data overestimate atmospheric CO2 levels, biasing long-term trends. Our results show that CO2 declined before and during Antarctic glaciation and support a substantial CO2 decrease as the primary agent forcing Antarctic glaciation, consistent with model-derived CO2 thresholds.