Climatic and environmental shifts have had profound impacts on faunal and floral assemblages globally since the end of the Miocene. We explore the regional expression of these fluctuations in southwestern Europe by constructing long-term records (from ~11.1 to 0.8 Ma, late Miocene-middle Pleistocene) of carbon and oxygen isotope variations in tooth enamel of different large herbivorous mammals from Spain. Isotopic differences among taxa illuminate differences in ecological niches. The δ(13)C values (relative to VPDB, mean -10.3 ± 1.1‰; range -13.0 to -7.4‰) are consistent with consumption of C3 vegetation; C4 plants did not contribute significantly to the diets of the selected taxa. When averaged by time interval to examine secular trends, δ(13)C values increase at ~9.5 Ma (MN9-MN10), probably related to the Middle Vallesian Crisis when there was a replacement of vegetation adapted to more humid conditions by vegetation adapted to drier and more seasonal conditions, and resulting in the disappearance of forested mammalian fauna. The mean δ(13)C value drops significantly at ~4.2-3.7 Ma (MN14-MN15) during the Pliocene Warm Period, which brought more humid conditions to Europe, and returns to higher δ(13)C values from ~2.6 Ma onwards (MN16), most likely reflecting more arid conditions as a consequence of the onset of the Northern Hemisphere glaciation. The most notable feature in oxygen isotope records (and mean annual temperature reconstructed from these records) is a gradual drop between MN13 and the middle Pleistocene (~6.3-0.8 Ma) most likely due to cooling associated with Northern Hemisphere glaciation.