We use the carbon and oxygen stable isotope content of mammalian tooth enamel to evaluate ancient biomes, habitat partitioning, and water use among 12 ungulate taxa from the White River Group in Nebraska. The White River Group spans an interval of climate change from the upper Eocene to the lower Oligocene when global climate cooled in a heterogeneous fashion. We test whether changes in biome structure or mammalian ecology occurred during this interval. We find that mean δ13C values for both Eocene and Oligocene faunas are consistent with dry, open biomes (e.g., woodland-savanna or woody scrubland). A significant increase in mean faunal δ13C values (+0.4‰) occurs in the lower Oligocene, primarily from the loss of individuals feeding in wetter–denser parts of the ecosystem. In the upper Eocene, Megacerops sp. (brontothere) and Perchoerus probus (peccary) have low mean δ13C values consistent with feeding in wetter, denser habitats (presumably riparian), whereas significantly higher mean values in the cursorial horse, Mesohippus spp., and the clawed oreodont, Agriochoerus antiquus, are consistent with feeding in more open, drier habitats. The extinctions of Megacerops sp. and Trigonias sp., a rhinocerotid with relatively low δ13C values, along with a decrease in the abundance of P. probus and other taxa, suggests reduction in the extent of wet habitats in the Oligocene. The rare tapir Colodon sp. is the only taxon sampled with a mean δ13C value low enough to indicate consistent feeding in a wet habitat in the Oligocene. However, range-through taxa occupy the same relative positions in isotopic space in both the Eocene and Oligocene faunas suggesting stability in drier habitats. A significant increase also occurs in mean faunal δ18O values (+1.0‰) in the Oligocene, primarily from the loss or reduction in abundance of water dependent species. Variance in δ18O values increases significantly in the Oligocene, which is consistent with greater water stress in plants from drier conditions. Oxygen values increase in A. antiquus (+0.9‰), the most aridity-sensitive species in both faunas, as expected for drier conditions, but values in another aridity-sensitive taxon (Mesohippus spp.) are virtually unchanged. Together, δ13C and δ18O enamel values generally suggest a shift to drier conditions in the early Oligocene although δ13C values yielded more consistent results. The lack of a large negative shift in δ18O values, expected with a decrease in mean annual temperature, suggests that any temperature changes in the early Oligocene were relatively small in the continental interior of North America.