This research presents the individual amino acid δ13C values in bone collagen of humans (n = 9) and animals (n = 27) from two prehistoric shell midden sites in Korea. We obtained complete baseline separation of 16 of the 18 amino acids found in bone collagen by using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). The isotopic results reveal that the humans and animals in the two sites had similar patterns in essential amino acids (EAAs) and non-essential amino acids (NEAAs). The EAA and NEAA δ13C values in humans are intermediate between those in marine and terrestrial animals. However, the threonine δ13C values in humans and animals measured in this study are more highly enriched than those of other amino acids. At both sites, all amino acids in marine animals are 13C-enriched relative to those of the terrestrial animals. The isotopic evidence suggests that the Tongsamdong human had EAAs and NEAAs from marine food resources, while the Nukdo humans mainly had EAAs from terrestrial food resources but obtained NEAAs from both terrestrial and marine resources. The δ13C isotopic differences in amino acids between marine and terrestrial animals were the largest for glycine (NEAA) and histidine (EAA) and the smallest for tyrosine (NEAA) and phenylalanine (EAA). In addition, threonine among the EAAs also had a large difference (∼8‰) in δ13C values between marine and terrestrial animals, and has the potential to be used as an isotopic marker in palaeodietary studies. Threonine δ13C values were used in conjunction with the established Δ13CGlycine–phenylalanine values and produced three distinct dietary groups (terrestrial, omnivorous, and marine). In addition, threonine δ13C values and Δ13CSerine–phenylalanine values were discovered to separate between two dietary groups (terrestrial vs. marine), and these δ13C values may provide a potential new indicator for investigating the distinction between marine and terrestrial protein sources in human diets.