Stable isotope analysis of animal tissue samples is increasingly used to study the trophic ecology of target species. The isotopic signatures respond to the type of diet, but also to the environmental conditions of their habitat. In the case of omnivorous, seasonal or opportunistic feeding species, the interpretation of isotopic values is more complex, as it is largely determined by food selection, either due to individual choice or because of availability. We analysed C and N isotopes in brown bear (Ursus arctos) hair from four isolated populations of south-western Europe (Cantabrian, Pyrenees, Central Apennines and Alpine) accounting for the geographical and climatic differences among the four areas. We found inter-population differences in isotopic signatures that cannot be attributed to climatic differences alone, indicating that at least some bears from relatively higher altitude populations experiencing higher precipitation (Pyrenees) show a greater consumption of animal foods than those from lower altitudes (Cantabrian and Apennines). The quantification of isotopic niche space using Layman’s metrics identified significant similarities between the Cantabrian and Central Apennine samples that markedly differ from the Pyrenean and Alpine. Our study provides a baseline to allow further comparisons in isotopic niche spaces in a broad ranged omnivorous mammal, whose European distribution requires further conservation attention especially for southern isolated populations.