A pronounced late Miocene cooling (LMC) from 7 to 5.7 Ma has been documented in extratropical and tropical sea surface temperature records, but to date, available proxy evidence has not revealed a significant pCO2 decline over this event. Here, we provide a new, high resolution pCO2 proxy record over the late Miocene cooling based on alkenone carbon isotopic fractionation (εp) measured in sediments from the South Atlantic at ODP Site 1088. We apply a recent proxy calibration derived from a compilation of laboratory cultures, which more accurately reflects the proxy sensitivity to pCO2 changes during late Quaternary glacial-interglacial cycles, together with new micropaleontological proxies to reconstruct past variations in algal growth rate, an important secondary influence on the εp. Our resulting pCO2 record suggests a 2-3 fold decline over the late Miocene Cooling and confirms a strong coupling between climate and pCO2 through the late Miocene. Within this long-term trend are pCO2 variations on sub-myr timescales that may reflect 400kyr long-eccentricity cycles, in which pCO2 minima coincide with several orbital scale maxima in published high-resolution benthic 𝛿18O records. These may correspond to ephemeral glaciations, potentially in the Northern Hemisphere. Our temperature and planktonic 𝛿18O records from Site 1088 are consistent with substantial equatorward movement of Southern Ocean frontal systems during the LMC. This suggests that potential feedbacks between cooling, ocean circulation and deep ocean CO2 storage may warrant further investigation during the LMC.