Middle Miocene mammals are known from similar to15 million-year-old sediments exposed along the Panama Canal of Central America, a region that otherwise has an exceedingly poor terrestrial fossil record. These land mammals, which represent a part of the ancient terrestrial herbivore community, include an oreodont Merycochoerus matthewi, small camel-like protoceratid artiodactyl Paratoceras wardi, two horses Anchitherium clarencei and Archaeohippus sp., and two rhinos Menoceras barbouri and Floridaceras whitei. Bulk and serial carbon and oxygen isotope analyses of the tooth enamel carbonate allow reconstruction of the ancient climate and ecology of these fossil mammals. Ancient Panama had an equable climate with seasonal temperature and rainfall fluctuations less than those seen today. The middle Miocene terrestrial community consisted predominantly, or exclusively, of C-3 plants, i.e., there is no evidence for C-4 grasses. Statistically different mean carbon isotope values for the mammalian herbivores indicate niche partitioning of the C-3 plant food resources. The range of individual carbon isotope analyses, i.e., delta(13)C from -15.9 to -10.1parts per thousand, indicates herbivores feeding on diverse plants from different habitats with extrapolated delta(13)C values of -29.9 to -24.2parts per thousand, possibly ranging from dense forest to more open country woodland. The ecological niches of individual mammalian herbivore species were differentiated either by diet or body size.