The Antarctic Ice Sheet (AIS) expansion and global cooling during the Mid-Miocene Climate Transition (MMCT) is thought to be closely linked to marine carbon cycle changes. However, how the marine carbon cycle interacted with the rest of the climate system during this period remains elusive. Here, we reconstruct surface-water CO2 and intermediate-depth seawater carbonate chemistry from the Southern Ocean during the MMCT. We show that a marked surface-water CO2 rise in the Southern Ocean, accompanied by carbon loss from the intermediate depths, coincided with AIS retreat and surface Southern Ocean warming within the MMCT. The release of CO2 from the intermediate depths to the surface ocean was likely caused by the northward shift of the Southern Ocean fronts and possibly strengthening of the Southern Ocean overturning circulation. Southern Ocean circulation reorganization, triggered by AIS expansion and global cooling, was able to transiently interrupt the transition of the Earth’s climate into a cooler state during the MMCT.