Neogene strata in the Meade Basin (southwest Kansas) preserve numerous superposed calcareous paleosols in sections that range in age from the Clarendonian North American Land Mammal Age (NALMA; 12.0–9.0 Ma, early late Miocene) to the early Irvingtonian NALMA (ca. 2.5–ca. 1 Ma, early Pleistocene). The carbon isotope compositions (δ13C relative to Vienna Peedee belemnite [VPDB]) of pedogenic carbonates from these sections record the protracted regional increase in the abundance of grasses using the C4 photosynthetic pathway over this interval, with the first appearance of a modern-like grassland ecosystem having >70% C4 biomass around 1.3 Ma. We use the stable oxygen isotope composition (δ18O relative to Vienna standard mean ocean water [VSMOW]) of 194 paleosol carbonates from 19 measured sections to reconstruct the climatic conditions in the Meade Basin during the rise of C4 grasses to ecological dominance in the region. Pedogenic carbonate δ18O values are sensitive to soil temperature and the δ18O of soil water, and hence they are a paleoclimate proxy. Carbonate δ13C values do not exhibit consistent trends in relation to δ18O values, indicating no consistent relationship between short-term climatic conditions in terms of temperature or aridity and the abundance of C4 biomass. Mean carbonate δ18O values within biostratigraphic intervals decrease from the Clarendonian (25.3‰ ± 0.72‰) to the early and middle Blancan (21.8‰ ± 0.87‰ and 22.1‰ ± 0.69‰, respectively), and they also exhibit a decreasing trend in the late Blancan–early Irvingtonian, from ∼25‰ to ∼21‰. The increase in δ18O values between the end of the middle Blancan and the beginning of the late Blancan–early Irvingtonian sections could reflect the onset of Northern Hemisphere glaciation and a change in the isotope composition of the hydrosphere due to increased ice volume. The long-term trend in δ18O values suggests that C4 biomass increased coincident with some combination of decreasing temperature, increasing proportion of winter precipitation recharge of soil water, and/or increasing soil moisture. Thus, the ecological dominance of C4 grasses in the region today does not seem to be linked to warmer temperatures or increased aridity.