AbstractOn Hokkaido, Japan, the Ezo wolfCanis lupus hattai, an apex predator, became extinct at the end of the 19th century owing to human activities. Top predators often have an important role in their ecosystems, yet we have no scientific information on the feeding habits of the Ezo wolf. We performed carbon and nitrogen stable isotope analysis and radiocarbon dating of specimens of the wolf (n = 7) and its prey species and estimated the components of the wolves’ diet using an isotope mixing model. Radiocarbon dating suggested that most of the wolves examined came from different populations or generations. The mean stable isotope ratios of the wolves were −19.5 ‰ (±1.9 ‰sd) forδ13C and 8.7 ‰ (±2.6 ‰sd) forδ15N. The discrimination‐corrected isotopic ratios of five of the seven wolves were almost the same as those of Sika deer at the same sites. In contrast, those of two wolves had clearly higher isotopic values than those of deer, suggesting that these wolves depended partly on marine prey such as salmon and marine mammals. Thus, Ezo wolves had similar ecological roles to Canadian grey wolves, and were a second subspecies shown to have fed on a marine diet, in addition to the ‘coastal wolves’ of British Columbia.