Nitrogen isotopes are widely used to study the trophic position of animals in modern food webs; however, their application in the fossil record is severely limited by degradation of organic material during fossilization. In this study, we show that the nitrogen isotope composition of organic matter preserved in mammalian tooth enamel (δ15Nenamel) records diet and trophic position. The δ15Nenamel of modern African mammals shows a 3.7‰ increase between herbivores and carnivores as expected from trophic enrichment, and there is a strong positive correlation between δ15Nenamel and δ15Nbone-collagen values from the same individuals. Additionally, δ15Nenamel values of Late Pleistocene fossil teeth preserve diet and trophic level information, despite complete diagenetic loss of collagen in the same specimens. We demonstrate that δ15Nenamel represents a powerful geochemical proxy for diet that is applicable to fossils and can help delineate major dietary transitions in ancient vertebrate lineages.