Trophic position is a fundamental characteristic of animals, yet it is unknown in many extinct species. In this study, we ground-truth the ¹⁵N/¹⁴N ratio of enameloid-bound organic matter (δ¹⁵N_EB) as a trophic level proxy by comparison to dentin collagen δ¹⁵N 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 δ¹⁵N_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. δ¹⁵N_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.