Using long-term diet reconstructions spanning the past one million years, we contrast hypotheses that biotic interactions versus physical environmental changes are primary drivers of evolutionary turnover in mammals. We use stable carbon (δ13C) and oxygen (δ18O) isotope ratios in tooth enamel carbonate to trace herbivore niche shifts through the Late Quaternary Land Mammal Ages (LMAs) of grassland savannas in the South African interior (Cornelian-1.0 to 0.6 Ma; Florisian-500 to 10 ka; and Holocene/modern). Data reveal niche separation amongst closely related coeval taxa, and dispersals through time into empty niche spaces following extinctions. This suggests a primary role of competitive exclusion and niche displacement for speciation and extinctions in these early grassland environments. However, niche changes through time show a similar trend in many taxa, entailing increased δ13C (elevated C4 grass consumption) from the Cornelian to the Florisian, and from the Florisian to the Holocene/modern, and elevated δ18O in Holocene/modern taxa that reflect global aridification around the terminal Pleistocene. Commonality in isotopic trends implies universal environmental forcing of ecological, and ultimately macroevolutionary, turnover. Yet some taxa shift from a mixed C3/C4 diet in the Florisian to a near-pure C3 diet today. Indeed, we find that while δ13C data are normally distributed for Cornelian fossils, non-normal distributions characterize more recent time intervals. Such distributions are in line with the bimodal distribution of δ13C and diet in contemporary African ungulates. Thus, while environmental forcing did not, by necessity, lead to increases in C4 intake, the results show changes from mixed to more specialized diets. We propose that this niche specialization was a function of long-term exposure to C4 grasslands, consistent with predictions that relatively high metabolic demands of C4 grazing in subtropical environments forced the differentiation of herbivores into one of two highly specialized feeding niches, i.e. C3 browsing or C4 grazing.