The Desmostylia, an extinct order of mammals related to sirenians and proboscideans, are known from the late Oligocene to late Miocene of the North Pacific. Though often categorized as marine mammals on the basis of fossil occurrences in nearshore deposits, reconstructions of desmostylian habitat and dietary preferences have been somewhat speculative because morphological and sedimentological information is limited. We analyzed the carbon, oxygen, and strontium isotope compositions of enamel from Desmostylus and co-occurring terrestrial and marine taxa from middle Miocene sites in California to address the debate surrounding desmostylian ecology. The δ13C value of tooth enamel can be used as a proxy for diet. Desmostylus had much higher δ13C values than coeval terrestrial or marine mammals, suggesting a unique diet that most likely consisted of aquatic vegetation. Modern aquatic mammals tend to exhibit lower variability in δ18O values than terrestrial mammals. Both fossil marine mammals and Desmostylus exhibited low δ18O variability, suggesting that Desmostylus spent a large amount of time in water. Finally, the Sr isotope composition of marine organisms reflects that of the ocean and is relatively invariant when compared with values for animals from land. Sr isotope values for Desmostylus were similar to those for terrestrial, rather than marine, mammals, suggesting Desmostylus was spending time in estuarine or freshwater environments. Together, isotopic data suggest that Desmostylus was an aquatic herbivore that spent a considerable portion of its life foraging in estuarine and freshwater ecosystems.