Shallow-water (<30 m) coral-reef fishes are under threat from multiple environmental and anthropogenic impacts. Typically spatially removed from such impacts, mesophotic coral ecosystems (MCEs) at intermediate depths (30-150 m) may serve as refugia for these fishes. In the main Hawaiian Islands, efforts to protect and manage coral-reef fisheries are underway. However, the inclusion of MCEs within management plans has been limited by a lack of information about the trophic structure of resident fish communities. Because of physical environment changes associated with increasing depth, we hypothesized that the diets of shallow and mesophotic fish would differ, with mesophotic fish relying less on benthic macroalgae and more on higher trophic-level prey. To address these possible ecological dietary changes, we examined the bulk tissue nitrogen and carbon isotopic compositions of 319 samples of reef fish from shallow and mesophotic depths, further analyzing the amino acid nitrogen isotopic composition of 81 samples. Significant differences were found in the bulk isotopic compositions of omnivores and benthic invertivores, whereas planktivores showed overlap between depths. Results of compound-specific isotopic analyses of amino acids indicated slightly but significantly higher trophic positions of mesophotic benthic invertivores compared to shallow-water members. Ecosystem models need to reflect the differences in food webs between shallow and mesophotic habitats, and results here place important constraints on outputs and assumptions for Hawaiian coral-reef habitats and potentially other reef tracts. Our results will allow better understanding of mesophotic fish ecology and stress the importance of MCE research in understanding trophic connectivity.