Glacial–interglacial changes in atmospheric CO2 are generally attributed to changes in seawater carbon chemistry in response to large-scale shifts in the ocean’s biogeochemistry and general circulation. The Southern Ocean currently takes up more CO2 than any other and it is likely to have played a crucial role in regulating past atmospheric CO2. However, the physical, biological and chemical variables that control ocean–atmosphere CO2 exchange during glacial–interglacial cycles are not completely understood. Here we use boron isotopes and carbon isotopes in planktonic foraminifera and an alkenone-based proxy of temperature to reconstruct seawater pH and CO2 partial pressure in sub-Antarctic surface waters south of Tasmania over the past 25,000 years, and investigate the mechanisms that regulate seawater CO2. The new record shows that surface waters in this region were a sink for atmospheric CO2 during the Last Glacial Maximum. Our reconstruction suggests changes in the strength of the biological pump and the release of deep-ocean CO2 to surface waters contributed to the last deglacial rise in atmospheric CO2. These findings demonstrate that variations in upwelling intensity and the distribution of Southern Ocean water masses in this sector played a key role in regulating atmospheric CO2 during the last glacial–interglacial cycle.