The extinction of megafauna in the Americas was among the most severe worldwide. In both North and South America, human hunting and the ecological effects of climate change were attributed as the leading causes of extinction. We hypothesize that megamammal populations were not adaptively vulnerable as has been proposed previously and that different megafauna species responded differently to climate and humans. To test this hypothesis, we used stable isotope analysis (delta C-13, delta O-18) of mineral phase bioapatite from megamammal remains from archaeological and paleontological sites located in the Argentine Pampas (Buenos Aires province) and covering the last similar to 16-11 cal ka BP, the time bin of the arrival and first human occupations in the region. We then computed Bayesian mixing models and reconstructed the isotopic niches of different megamammal taxa. We reconstruct aspects of the megamammal community structure during the initial period of human occupation and set qualitative and quantitative bases to predict potential shifts in their isotopic niches. Our results show that the megafauna of the Argentine Pampas during the latest Pleistocene had different habitats and food preferences, spanning a wide range of trophic categories. These findings are essential to evaluate how different megamammal species responded to human and climatic pressures leading to their eventual extinction.