The oxygen isotope composition of fossil fish teeth, a paleo– upper ocean temperature proxy exceptionally resistant to diagenetic alteration, provides new insight on the evolution of the low- to middle latitude thermal gradient between the middle Cretaceous climatic optimum and the cooler latest Cretaceous period. The new middle Cretaceous low to middle latitude thermal gradient agrees with that previously inferred from planktonic foraminifera 18O recovered from Deep Sea Drilling Project and Ocean Drilling Program drilling sites, although the isotopic temperatures derived from 18O of fish teeth are uniformly higher by ~3–4 °C. In contrast, our new latest Cretaceous thermal gradient is markedly steeper than those previously published for this period. Fish tooth 18O data demonstrate that low- to middle-latitude thermal gradients of the middle Cretaceous climatic optimum and of the cooler latest Cretaceous are similar to the modern one, despite a cooling of 7 °C between the two periods. Our new results imply that no drastic changes in meridional heat transport are required to explain the Late Cretaceous climate. Based on climate models, such a cooling without any change in the low to middle latitude thermal gradient supports an atmospheric CO2 decrease as the primary driver of the climatic evolution recorded during the Late Cretaceous.