Compound-specific isotope analysis of individual amino acids (AA) is a rapidly growing tool in ecological studies to assess diet and trophic position (TP) in both modern and ancient foodwebs. We conducted the first controlled feeding study examining δ 15 N values in AAs in a marine mammal (harbor seal Phoca vitulina). The pattern of δ 15 N variation among AAs in seals was similar to that observed in other heterotrophs, although exceptions were found with proline and threonine. However, many δ 15 N changes with trophic transfer were very different than those reported for zooplankton and other lower TP marine consumers. In particular the measured trophic enrichment factor (TEF) now broadly used for TP estimation (TEF Glu-Phe) was much lower in harbor seals (~4.3 ‰) than the current commonly applied value (~7.5 ‰). Recently published data on wild marine birds (penguins) and elasmobranchs (stingrays) suggests that similar, low TEF values may also be characteristic of these taxa. Together, these data imply that marine mammals and other higher animals have different, but also diagnostic, changes in δ 15 N-AA with trophic transfer vs. organisms examined in previous feeding studies (e.g. zooplankton, bony fish and mollusks), possibly due to dietary protein content, trophic position, and/or form of nitrogen excretion (urea vs. ammonia). Therefore, we propose that for marine mammals, a multi-TEF calculation is required to account for variations of TEF between animals within a food web, and we demonstrate that this approach can predict accurate TP estimates for harbor seals. These results also have significant implication for the application of compound-specific isotope analysis of AAs on terrestrial ecology and trophic structure.