The Pampa in subtropical Brazil (State of Rio Grande do Sul) is the only area of South America known so far where fossils of the Pleistocene giant megatheriid sloths Megatherium americanum, characteristic of subtropical-temperate areas, and Eremotherium laurillardi, widespread in the tropical zone, were discovered in the same deposits (Pessegueiro Creek and Chuí Creek), but it is not clear whether this co-occurrence is a product of taphonomic mixture, or co-existence, which would imply niche partitioning. In order to understand their paleoecology and reconstruct the associated paleoenvironments, dentin samples of both megatheriids from the two sites were analyzed for their carbon (δ13C) and oxygen (δ18O) isotope ratios. The δ13C values of Megatherium indicate mixed diet of C3-C4 plants with higher content of the latter in Pessegueiro Creek, whereas the δ13C values of Eremotherium indicate C3-dominated diets, the more negative value in Pessegueiro Creek possibly related to the canopy effect. The δ18O of Eremotherium points to 18O-depleted water sources, possibly also influenced by the diet, whereas Megatherium ingested 18O-enriched water. The results show that co-existence of both megatheriids would have been ecologically possible, and that the Pampa was occupied by open grasslands/woodlands, with closed forests in Pessegueiro Creek. The δ13C of Eremotherium from intertropical Brazil indicate a more generalist habit than Megatherium from subtropical Brazil and Argentina, which probably facilitated its dispersion from the tropics up to subtropical areas along two different routes, one along the coastal plain, and the other along the Paraná River Basin across central South America, following the southward expansion of riparian forests during warmer stages. The reduction of those forests during intervening cold stages possibly led to its disappearance in southern Brazil. Keywords: Quaternary, paleobiogeography, stable isotopes, Pampa, coastal plain, megafauna.