Bison (Bison bison) are one of the few terrestrial megafauna to survive the transition into the Holocene and provide a unique opportunity to study a species on a broad spatiotemporal scale. Today, bison are primarily managed in small and isolated herds with little known about their ancestral ecology. We studied the carbon and nitrogen isotopes of Northern Great Plains bison from the terminal Pleistocene and throughout the Holocene to gain insight into their paleoecology. This time span is contemporary with the first population bottleneck experienced by bison at the end of the Pleistocene and includes the second bottleneck which occurred in the late 19th century. Results were compared with modern bison herd isotopic values from Theodore Roosevelt National Park (TRNP). Patterns of isotopic variation found in bison over time indicate significant (δ13C p = 0.0008, δ15N p = 0.002) differences in diet composition and correlate with climate throughout the Holocene. Isotopic relationships described here reveal the plasticity of ancient bison in unrestricted rangelands during periods of climatic fluctuations. Managers at TRNP and elsewhere should pursue opportunities to expand bison range to maximize forage opportunities for the species in the face of future environmental change.