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The expansion of C-4 grassland in the Neogene, starting from 4 to 9 million years ago, is thought to be caused by decreasing CO2 concentration, increasing aridity, or a combination of both. Oxygen isotopes from paleoproxies show a decreasing trend and an increasing variability from mid-Miocene to Pleistocene in the Southern Great Plains of the United States, where carbon isotopes indicate an overall increase of C-4 plants during the same time period. Using isotope-enabled General Circulation Model (GCM) simulations, here I explore the causes of the long-term trend and variability of oxygen isotopes, ultimately to understand the climatic condition that is associated with the C-4 grassland expansion. Climate model simulations were performed varying the orbital configuration, CO2 concentration, and vegetation coverage. Model results show that the isotopic composition of winter precipitation varies more than that of summer precipitation and depends strongly on temperature, or more precisely on the contribution of snow to total precipitation. I conclude that the long-term isotopic composition of precipitation decreases because of gradual cooling and varies more because of lower and more variable cold season temperature in a colder climate in response to fluctuating orbital configuration. This study implies that C-4 grassland in the Southern Great Plains region expanded along with global cooling, possibly resulting from the decreasing atmospheric CO2 concentration.