The global climate has been gradually cooling over the Cenozoic and is punctuated by the intensification of Northern Hemisphere Glaciation (NHG) from the latest Pliocene to earliest Pleistocene (∼3.1–2.5 millions of years ago, Ma). A decline of atmospheric CO2 is supposed as a prerequisite for the NHG, but the associated carbon-cycle processes remain elusive. Here we combine foraminiferal records of neodymium isotope and boron-calcium ratio, and simulations of an Earth system model, to investigate changes in the water-mass composition and carbonate-ion concentration of the deep Pacific Ocean during the NHG. Our proxy records have revealed a significant expansion of southern-sourced waters with increased respired carbon storage into the deep Pacific during the NHG. These changes may be explained by strengthened deep-water formation and biological-pump efficiency in the Southern Ocean due to Antarctic sea-ice growth, as suggested by our model experiments and evidence from the Sub-Antarctic region. These results provide key clues for quantifying the role of the dissolved inorganic carbon content of deep Pacific waters in modulating atmospheric CO2 during the NHG.