Abstract Shortfin mako shark, Isurus oxyrinchus , is listed as an endangered species with declining global population. Thus, studies regarding its biology and ecology are important to recommend fishery management and conservation measures. This study aimed to determine the diet composition and feeding habits of I. oxyrinchus in Ecuadorian waters. Samples were obtained from Santa Rosa fishing port (Ecuador). The total length (L T ), sex and sexual characteristics were recorded, and stomach contents were collected. A total of 142 individuals were recorded, comprising 81 females (104–295 cm L T ) and 61 males (127–245 cm L T ). A total of 24 prey species were identified, including crustaceans, cephalopods, teleosts and cetaceans. According to the Prey-Specific Index of Relative Importance (PSIRI), the main prey taxa were the ommastrephid squid, Dosidicus gigas (42.57%) and Sthenoteuthis oualaniensis (21.04%), followed by fish from the family Hemiramphidae (11.85%). Isurus oxyrinchus is a specialist predator that preferred a low number of prey ( Bi = 0.25), both by sex ( Bi ; females = 0.29 and males = 0.34) and life stages ( Bi ; juveniles = 0.27 and adults = 0.37). The trophic overlap was medium for sexes ( J = 0.54) and biological cycle phases ( J = 0.42). Trophic level ( TL k ) was 4.47, indicating that I. oxyrinchus is a tertiary predator. This information will help in fisheries management based on an ecosystem approach, where this species fulfils an ecological role, and its interactions with other species allow us to understand how the flow of nutrients and energy occurs within an ecosystem.