The analysis of stable isotopes on fossil mammals has become a widely used tool for understanding the paleoecology and paleodiet of these organisms. In this work, we study the stable isotope composition of collagen (δ¹³C and δ¹⁵N) and bioapatite (δ¹³C and δ¹⁸O) of fossil bones from Arroyo del Vizcaíno, a fossiliferous site in southern Uruguay dated to ∼32 ka cal BP. Fourteen taxa were analyzed: the ground sloths Lestodon armatus, Glossotherium robustum, Mylodon darwinii, Valgipes bucklandi and Nothrotheriops sp.; the glyptodonts Glyptodon reticulatus, Panochthus tuberculatus, and Doedicurus clavicaudatus; the equids Hippidion principale and Equus neogeus; the proboscidean Notiomastodon platensis; the notoungulate Toxodon platensis; the saber-tooth felid Smilodon populator; and an indeterminate cervid. In general, the percentage of nitrogen in collagen and the C:N ratio were within the limits expected for collagen preservation in fossil bone. The δ¹³C and δ¹⁵N results of the herbivores indicated that most taxa consumed high proportions of C₃ plants in open, relatively dry environments, but significant variations were observed. Furthermore, relatively high values of δ¹⁸O in bone bioapatite, which approximately tracks local drinking water, indicated low precipitation and/or high evaporation. Our results show that the herbivorous taxa present at the site covered a considerable range of the grazer-browser spectrum and support the existence of niche partitioning among closely related taxa. Overall, these kinds of approaches are indispensable to better understand how these communities thrived during the Pleistocene in the region, supporting an outstanding number of giant species, before their extinction at the Pleistocene-Holocene transition.