Species invasions pose a serious threat to native biodiversity and ecosystems. However, quantifying the impacts of invasive species has proven problematic. In this study, we quantified the trophic changes in freshwater food webs invaded by tilapia, using an extensive stable isotope dataset to compare uninvaded and invaded rivers downstream of the Pearl River, China. The trophic position of the widely distributed and locally economically important piscivorous culter fish (Erythroculter recurviceps), mandarin fish (Siniperca kneri), and catfish (Pelteobagrus fulvidraco and Pelteobagrus vachelli) decreased significantly in the invaded river compared to the uninvaded river. Our analysis indicated that the decrease in tropic position of these piscivorous fishes reflected a major reduction in the proportion of prey fish biomass as a result of tilapia invasion. Stable isotope mixing models (SIMMs) indicated that small fish in the diet of culter fish from the reference river (32.7% small fish, 17% zooplankton) were replaced by lower trophic level zooplankton prey in the invaded river (35.7% zooplankton, 25.4% small fish), due to the presence of tilapia. Small fish in the diet of mandarin fish in the reference river (46.2% small fish, 10.5% aquatic insects) was replaced by lower trophic level aquatic insect prey in the invaded river (20.3% aquatic insects, 29.9% small fish). Fish eggs in the diet of catfish from the reference river (25.0% fish eggs, 25.2% aquatic insects) were replaced by aquatic insects at a lower trophic level in the invaded river (43.5% aquatic insects, 4.8% fish eggs). The results of this study contributed to a growing body of evidence, showing that tilapia could modify trophic interactions, which had severe consequences in invaded ecosystems.