We use the carbon isotope composition of paleosols to reconstruct the history of C4 biomass on the Great Plains from ca. 23 to 1 Ma. The proportion of C4 biomass was uniform and moderate (12%–34%) throughout the Miocene, increased between 6.4 and 4.0 Ma, and reached modern levels by 2.5 Ma. Ecological changes in Great Plains ungulates preceded the increase in C4 biomass. The contrasts in the paleosol and ungulate records may indicate initial development of C3 grasslands after the middle Miocene or a greater role for ecological interactions within communities in structuring ungulate faunas. Contrasts in paleosol records from different continents point to regional rather than global controls on the evolution of C4 grasslands.