Trophic position is a fundamental characteristic of animals, yet it is unknown in many extinct species. In this study, we ground-truth the 15 N/ 14 N ratio of enameloid-bound organic matter (δ 15 N EB ) as a trophic level proxy by comparison to dentin collagen δ 15 N and apply this method to the fossil record to reconstruct the trophic level of the megatooth sharks (genus Otodus ). These sharks evolved in the Cenozoic, culminating in Otodus megalodon , a shark with a maximum body size of more than 15 m, which went extinct 3.5 million years ago. Very high δ 15 N EB values (22.9 ± 4.4‰) of O. megalodon from the Miocene and Pliocene show that it occupied a higher trophic level than is known for any marine species, extinct or extant. δ 15 N EB also indicates a dietary shift in sharks of the megatooth lineage as they evolved toward the gigantic O. megalodon , with the highest trophic level apparently reached earlier than peak size.