Carbon isotope ratios (δ13C values) of herbivores reflect the δ13C values of dietary plants, and the δ13C values of grazers (animals that consume >90% grass) reflect the local abundance of C3 versus C4 grasses. Because grassland C3/C4 ratios correlate with climate, the δ13C of fossil grazers may serve as a proxy for reconstructing paleoclimates and paleovegetation patterns. However, the accuracy of environmental reconstructions based on herbivore δ13C values is often uncertain, because the relationship between the δ13C of many animals and the abundance of C4 and C3 grasses has not been precisely quantified. We analyzed the δ13C of tooth enamel carbonate from modern bison (Bison bison bison) from nine localities in the United States. The C4 grass biomass at these sites ranged from <1% to ∼95% of the total grass biomass. The mean δ13C of enamel for each population correlated well with the local abundance of C4 grasses and with variations in mean annual temperatures. The variability of enamel δ13C values did not differ among habitats and was not correlated with the abundance of C4 grasses. These results demonstrate that analyses of the δ13C values of fossil bison can be used as a quantitative proxy for reconstructing grassland C3/C4 ratios and paleotemperatures, and they will serve as a baseline for interpreting the δ13C of fossil bison and other large herbivores in North America.