Large pelagic fishes are top predators inhabiting the water column in the tropical and subtropical Pacific Ocean. They are highly migratory organisms, and for Ecuadorian fisheries, they also represent important economic resources. We sampled four shark species, Prionace glauca, Isurus oxyrinchus, Carcharhinus falciformis, and Alopias pelagicus, and three billfish species, Xiphias gladius,
... [Show full abstract] Istiophorus platypterus and Makaira nigricans, to understand their feeding ecology and trophic interactions through the use and combination of stomach content and stable isotope analyses. Results showed that δ13C values were similar among the seven pelagic predators (values ranged between -16.45‰ for M. nigricans and -16.73‰ for C. falciformis) suggesting that all of them exploit a similar marine area off the coast of Ecuador. δ15N stable isotope values differed among species (the lowest value was 13.83‰ for A. pelagicus and the highest value was 18.57‰ for P. glauca) suggesting a segregation in the use of the water column and/or in the diet. δ15N values, indicated that the consumption of cephalopods and fish determined habitat use and depredation in the ecosystem. The sharks I. oxyrinchus and P. glauca had high values of δ15N, and preyed mainly on cephalopods in comparison with the other species, which preyed mainly on fish. In addition to the common use of cephalopods as prey for large pelagic fish, our results indicate that these shark species segregate their diet.