The fossil record of marine microplankton provides insights into the evolutionary drivers which led to the origin of modern deep-water plankton, one of the largest components of ocean biomass. We use global abundance and biogeographic data, combined with depth habitat reconstructions, to determine the environmental mechanisms behind speciation in two groups of pelagic microfossils over the past 15 Myr. We compare our microfossil datasets with water column profiles simulated in an Earth system model. We show that deep-living planktonic foraminiferal (zooplankton) and calcareous nannofossil (mixotroph phytoplankton) species were virtually absent globally during the peak of the middle Miocene warmth. The evolution of deep-dwelling planktonic foraminifera started from subpolar–mid-latitude species, during late Miocene cooling, via allopatry. Deep-dwelling species subsequently spread towards lower latitudes and further diversified via depth sympatry, establishing modern communities stratified hundreds of metres down the water column. Similarly, sub-euphotic zone specialist calcareous nannofossils become a major component of tropical and sub-tropical assemblages during the latest Miocene to early Pliocene. Our model simulations suggest that increased organic matter and oxygen availability for planktonic foraminifera, and increased nutrients and light penetration for nannoplankton, favoured the evolution of new deep-water niches. These conditions resulted from global cooling and the associated increase in the efficiency of the biological pump over the last 15 Myr.