Stable isotope analysis of mammalian tooth enamel is a valuable method for examining resource partitioning in modern and ancient environments where there is a mixture of C3 and C4 plants. However, before 7 Ma North American ecosystems were composed predominantly of C3 plants, complicating isotopic assessment of resource partitioning. Study of modern African and North American ecosystems has shown that niche partitioning among mammals may be discerned in communities dominated by C3 plants, suggesting that a similar approach may work for ancient C3 ecosystems. Here, such analyses are applied to explore resource use and niche partitioning in two ancient C3-dominated communities, one from California and one from Florida. Each locality, Black Hawk Ranch (California) and the Love Bone Bed (Florida), occurs in Miocene deposits that accumulated prior to the rapid increase in C4 ecosystems 7 Myr ago. δ13C and δ18O values were obtained from the tooth enamel of eight species from Black Hawk Ranch, and 15 species from the Love Bone Bed. Results from the 197 bulk isotope samples showed significant differences in δ13C among taxa at the Love Bone Bed, but no significant differences were observed among taxa at Black Hawk Ranch. At both localities, equids generally have more positive δ13C values than co-occurring taxa, suggesting that equids occupied more open habitats, whereas antilocaprids, camelids, and proboscideans have more negative values, implying utilization of more closed communities. One result of note is the positive δ13C values of Pediomeryx (Yumaceras) hamiltoni from the Love Bone Bed, which suggests that P. (Y.) hamiltoni incorporated abundant fiber, possibly grass, in the diet similar to the horses from this locality. The lack of significant differences among taxa at Black Hawk Ranch may indicate a relatively homogeneous flora, or presence of abundant resources permitting niche overlap, whereas the opposite is implied by the presence of significantly different isotope values among taxa at the Love Bone Bed. The results from this study highlight the utility of isotopic techniques allowing discernment of resource partitioning in C3-dominated landscapes such as those that persisted for the millions of years before the rapid increase in C4 ecosystems that occurred during the late Miocene.