The diet of extinct giant Xenarthrans is a debated topic, especially for ground sloths, for which herbivory, insectivory, and carnivory through scavenging or active hunting have been suggested. In this study, stable carbon isotopic composition of collagen and carbonate fraction of well-preserved fossil bones was used as a tracer of trophic level. >200 modern mammal bones of species with a variety of diets were used as a reference dataset. The good preservation of the carbonate isotopic composition was checked using different diagenetic indicators and by using fossil carnivorous and herbivorous taxa from the same sites as test-taxa. The results on modern mammals indicate a clear distinction in the difference between the carbon isotopic composition of carbonate and collagen between faunivores (carnivores, insectivores, piscivores, omnivores) and herbivores. Using this framework, the results on the fossil megafaunal Xenarthrans indicate a herbivorous diet for both Glyptodontids and are also consistent with a herbivorous diet for giant ground sloths, including Megatherium. The hypothesis that Megatherium could be a cryptic meat-eater or an insectivore is not supported by the results obtained in the present study.