Large-bodied, semiaquatic herbivorous mammals have been a recurring component of most continental ecosystems throughout the Cenozoic. Identification of these species in the fossil record has largely been based on the morphological similarities with present-day hippopotamids, leading to the designation of this pairing of body type and ecological niche as the hippo ecomorph. These morphological characters, however, may not always be diagnostic of aquatic habits. Here, enamel δ13C and δ18O values from living hippopotamuses were examined to define an isotopic signature unique to the hippo ecomorph. Although δ13C values do not support unique foraging habits for this ecomorph, living and fossil hippopotamids typically have low mean δ18O values relative to associated ungulates that fit a linear regression (δ18Ohippopotamids = 0.96 ± 0.09·δ18Ofauna − 1.67 ± 2.97; r2 = 0.886, p < 0.001). Modeling of oxygen fluxes in large mammals suggests that high water-turnover rates or increased water loss through feces and urine may explain this relationship. This relationship was then used to assess the aquatic adaptation of four purported hippo ecomorphs from the fossil record: Coryphodon (early Eocene), Moeritherium and Bothriogenys (early Oligocene), and Teleoceras (middle–late Miocene). Only fossil specimens of Moeritherium, Bothriogenys, and large species of Coryphodon had δ18O values expected for hippo ecomorphs; δ18O values for Teleoceras and a small species of Coryphodon were not significantly different from those of the associated fauna. These results show that the mean δ18O value of fossil specimens is an effective tool for assessing the aquatic habits of extinct species.