This study aims at assessing resource and habitat use, niche occupation and trophic interactions from a stable isotope perspective on fossil mammals from the Argentine pampas during the Great American Biotic Interchange (GABI). We present stable isotope data of more than 400 samples belonging to 10 mammalian orders and spanning a temporal range from ~9.5 Ma to ~12 ky. Rodents, notoungulates and pilosians record an increase in the consumption of c 4 plants, whereas litopterns and cingulates show δ 13 c values that remain mostly within a c 3 -dominated diet. Our stable isotope data indicates that the expansion of c 4 vegetation opened up new niche opportunities, probably alleviating resource competition among endemic taxa. Gomphothere, equid and camelid δ 13 c records show a broad variability pointing to consumption of c 3 and mixed c 3 -c 4 vegetation. This flexible dietary behavior may have facilitated the successful settlement of immigrant groups in South America. In the case of carnivorous taxa, Late Miocene pre-GABI endemic sparassodonts consumed prey from C 3 environments, whereas immigrant carnivorans preferred prey from mixed C 3 -c 4 areas. Our research contributes to the study of the GABI from a different perspective as stable isotope records permit to characterize, from a (semi)quantitative standpoint, ecological traits within extinct fauna.Understanding the evolution of mammalian communities throughout the history of Earth, their resource and habitat use, niche occupation and trophic relationships play fundamental roles as these variables determine interaction among species, which ultimately trigger profound changes in community structure leading to modern faunal structure 1 .The Late Cenozoic fossil record of South America provides a unique natural laboratory to investigate faunal response in the context of changing biotic and abiotic forces 2 . Recent works on the tectonism, paleoceanography, paleobiogeography and paleobiology of the Isthmus of Panama have refined our knowledge of its final formation, although not without controversy and sometimes opposing views about its tempo and mode 3 . Despite debates about tectonic vs. ecological barriers to intercontinental faunal exchange, most workers accept that South America was mostly isolated from other continents for more than 50 Ma, from the Late Paleocene until regular terrestrial faunal exchange began following the establishment of the Panama corridor 4,5 .The Great American Biotic Interchange (GABI) occurred in pulses, reflecting intermittent connections due to sea level lowstands and glacial-interglacial climatic dynamics 4,6-8 . The first arrival of North American taxa into South America took place in the Late Miocene (Huayquerian) before the total establishment of the Panama corridor. Herald taxa were procyonids, cricetids and later on, tayassuids. Then, once the land bridge was completed