We integrated stomach content analysis (SCA) and stable isotope analysis (SIA) to understand ontogenetic niche shifts in the invasive freshwater fish, bluegill, Lepomis macrochirus, inhabiting the Yedang Reservoir in Korea. Based on the total length (TL), we classified L. macrochirus as small (23–57 mm), medium (61–99 mm), or large (100–163 mm). Across all study sites, the index of relative importance (IRI) of zooplankton was high for small individuals, whereas those of benthic macroinvertebrates were high for the medium and large groups. Isotopic niche width estimates based on carbon and nitrogen isotope ratios (δ space) also increased with growth, indicating an ontogenetic niche shift in L. macrochirus. In particular, the δ space and diet plasticity of large fish was higher in the littoral population, implying L. macrochirus are generalist feeders as adults. Individuals classified as small showed confined and constant δ space, regardless of habitat condition. Thus, together with the results on the significantly high IRI of zooplankton, these individuals seem to have strong specialistic feeding ecology. Our study demonstrates the applicability strength of combining SCA and SIA for ecological niche research by providing clear evidence of an ontogenetic niche shift in L. macrochirus and elucidates their feeding ecology.