The Neogene Period (23.03–2.58 Ma) underwent a long-term, relatively gradual cooling trend, culminating in the glacial-interglacial climate of the Quaternary. Palaeoclimate studies on the Neogene have provided important information for understanding how modern patterns of atmospheric and oceanic circulation developed, and how they may relate to wider environmental change. Here we use a newly created global database of Neogene dinoflagellate cysts (the Tertiary Oceanic Parameters Information System - TOPIS) to investigate how dinoflagellate cysts recorded the cooling of Neogene surface marine waters on a global scale. Species with warm and cold water preferences were determined from previously published literature and extracted from the database. Percentages of cold water species were calculated relative to the total number of species with known temperature preferences from each site and compared throughout the Neogene at differing latitudes. Overall, the percentage of cold water species increases gradually through the Neogene. This trend indicates a gradual global cooling that is comparable to that reported from other marine and terrestrial proxies. This also demonstrates the use of dinoflagellate cysts in determining temperature change on both extended temporal and wide geographical scales. The increase in the percentage of cold water species of dinoflagellate cysts recorded worldwide from the Early and Middle Miocene to the Late Pliocene indicates a global scale forcing agent on Neogene climate such as CO2.