Urban environments are novel alternative habitats for wildlife. Anthropogenic food resources such as supplemental feeding (SF) are usually abundant, stable across seasons, and sometimes rich in calories. Although anthropogenic food resources could modify the seasonality and composition of the diets of urban animals, previous studies have been limited to qualitative assessments based on stomach content and food remains. Our study compared diet seasonality and niche width between rural and urban populations of Eurasian red squirrels (Sciurus vulgaris) in Hokkaido, Japan, using direct observation and stable isotope analysis. We found that dietary seasonality was lower in urban populations than in rural populations. The frequency of seed use was higher throughout the year in urban populations than in rural populations, possibly because of excessive seeds by SF in urban squirrels. Additionally, female body weights of the urban population were higher than those of the rural population. However, this was not true for males. Because high body weight can increase reproductive success in females, it is feasible that diet modification could contribute to population growth in urban squirrels. However, no differences in male body weight were observed, possibly due to energy loss caused by intense male–male competition at feeding sites. Our study highlights the importance of studying the multidimensional effects of anthropogenic foods on wildlife.