Trophic position is a fundamental characteristic of animals, yet it is unknown in many extinct species. In this study, we ground-truth the15N/14N ratio of enameloid-bound organic matter (δ15NEB) 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 (genusOtodus). These sharks evolved in the Cenozoic, culminating inOtodus megalodon, a shark with a maximum body size of more than 15 m, which went extinct 3.5 million years ago. Very high δ15NEBvalues (22.9 ± 4.4‰) ofO. megalodonfrom the Miocene and Pliocene show that it occupied a higher trophic level than is known for any marine species, extinct or extant. δ15NEBalso indicates a dietary shift in sharks of the megatooth lineage as they evolved toward the giganticO. megalodon, with the highest trophic level apparently reached earlier than peak size.Nitrogen isotope ratios in fossil teeth place extinct megatooth sharks at the top of the marine food web.