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Incorporation of animal-based foods into early hominin diets has been hypothesized to be a major catalyst of many important evolutionary events, including brain expansion. However, direct evidence of the onset and evolution of animal resource consumption in hominins remains elusive. The nitrogen-15 to nitrogen-14 ratio of collagen provides trophic information about individuals in modern and geologically recent ecosystems (<200,000 years ago), but diagenetic loss of this organic matter precludes studies of greater age. By contrast, nitrogen in tooth enamel is preserved for millions of years. We report enamel-bound organic nitrogen and carbonate carbon isotope measurements of Sterkfontein Member 4 mammalian fauna, including seven Australopithecus specimens. Our results suggest a variable but plant-based diet (largely C₃) for these hominins. Therefore, we argue that Australopithecus at Sterkfontein did not engage in regular mammalian meat consumption.