Hominin relationships and their dispersal throughout Eurasia during the Early and Middle Pleistocene are highly debated. The relationships between Early and Middle Pleistocene hominins, like Homo antecessor and Homo erectus, and hominin species that dominate the Late Pleistocene fossil record, like Homo sapiens, Neanderthals and Denisovans, are unclear. Here, we obtain enamel proteomes from Homo antecessor (Atapuerca, Spain) and Homo erectus (Dmanisi, Georgia), two key fossil assemblages that have a central role in models of Pleistocene hominin morphology, dispersal, and divergence. We recover endogenous ancient proteomes from the Atapuerca and Dmanisi hominins. In addition, our proteomic data indicates that the sampled Homo antecessor molar derives from a male hominin through amelogenin protein sexing. Our data includes preserved in vivo phosphorylation and enamel proteome proteolytic digestion.