We used carbon and nitrogen stable isotope analyses to assess the relative contributions from pelagic and littoral energy sources to higher trophic levels in a lake ecosystem before and after a major food web perturbation. The food web structure of the lake was altered when the population sizes of the most abundant fish species (small perch, roach and bream) were reduced during an attempt to improve water quality by biomanipulation. Fish removal was followed by dense year classes of young fish, which subsequently increased the utilisation of pelagic resources. This was reflected as a decrease in relative energy contribution from littoral sources and also led to more distinct pelagic and littoral food chains after fish removal. Community metrics calculated from stable isotope data indicated increased trophic diversity and occupied niche area, and reduced trophic redundancy in the food web. However, only minor changes were observed in fish trophic positions, although roach and pike occupied slightly lower trophic positions after fish removal. Despite the Jyväsjärvi ecosystem becoming more dependent on pelagic energy after fish removals, the littoral energy contribution was still substantial, particularly to certain fish species. Hence, our results support recent arguments for the importance of benthic production in lake ecosystems. More generally, our results illustrate how large-scale perturbations of food web structure can alter energy flow patterns through an entire ecosystem.