The order Proboscidea includes extant elephants and their extinct relatives and is closely related to the aquatic sirenians (manatees and dugongs) and terrestrial hyracoids (hyraxes). Some analyses of embryological, morphological, and paleontological data suggest that proboscideans and sirenians shared an aquatic or semiaquatic common ancestor, but independent tests of this hypothesis have proven elusive. Here we test the hypothesis of an aquatic ancestry for advanced proboscideans by measuring ␦ 18 O in tooth enamel of two late Eocene proboscidean genera, Barytherium and Moeritherium, which are sister taxa of Oligocene-to-Recent proboscideans. The combination of low ␦ 18 O values and low ␦ 18 O standard deviations in Barytherium and Moeritherium matches the isotopic pattern seen in aquatic and semiaquatic mammals, and differs from that of terrestrial mammals. ␦ 13 C values of these early proboscideans suggest that both genera are likely to have consumed freshwater plants, although a component of C 3 terrestrial vegetation cannot be ruled out. The simplest explanation for the combined evidence from isotopes, dental functional morphology, and depositional environments is that Barytherium and Moeritherium were at least semiaquatic and lived in freshwater swamp or riverine environments, where they grazed on freshwater vegetation. These results lend new support to the hypothesis that Oligocene-to-Recent proboscideans are derived from amphibious ancestors.Barytherium ͉ Eocene ͉ Fayum ͉ Moeritherium ͉ Proboscidea T he elephants Elephas and Loxodonta (order Proboscidea) are the only living remnants of a major adaptive radiation whose origin can now be traced back to the earliest Eocene (Ϸ55 million years ago) in Africa (1). Genomic data place proboscideans within the placental mammalian superorder Afrotheria (2) and the more restricted supraordinal clade Paenungulata, which also contains the aquatic manatees and dugongs (order Sirenia) and terrestrial hyraxes (order Hyracoidea). Genetic evidence has thus far failed to resolve relationships among paenungulate orders (2, 3), but a recent analysis of genomic and morphological evidence provided weak support for a Proboscidea-Sirenia clade (Tethytheria) to the exclusion of Hyracoidea (4). A monophyletic Tethytheria has long been seen as the best explanation for available morphological evidence (5) and is key to the hypothesis [also based on developmental (6) and paleontological (5, 7) evidence] that the common ancestor of elephants and sea cows might have been at least semiaquatic.Eocene proboscideans were radically different from living elephants in their size, skeletal and dental morphology, and presumably many aspects of their ecology and behavior as well (5,(7)(8)(9)