Little is known about the ecology of round goby (Neogobius melanostomus), an invasive benthic fish, in the profundal zone of the Great Lakes. In April 2002–2005 we caught increasing numbers of round gobies with a bottom trawl in the 45–150 m depth range of southwestern Lake Ontario. In 2005, we examined gut contents of 30 round gobies from each of three depths, 55, 95, and 130 m, and qualitatively compared gut contents with density of benthic invertebrates determined by Ponar grabs. Round goby guts contained mostly Dreissena spp. and opposum shrimp, Mysis relicta (Mysis); the frequency of occurrence of dreissenids in guts decreased with depth, whereas the frequency of occurrence of Mysis in guts increased with depth. Abundance of these invertebrates in the environment followed the same pattern, although dreissenids of optimum edible size (3–12 mm) were still abundant (1,373/m²) at 130 m, where round gobies primarily consumed Mysis, suggesting that round gobies may switch from dreissenids to more profitable prey when it is available. Other food items were ostracods and fish, with ostracods generally eaten by smaller round gobies and fish eaten by larger round gobies. Occurrence and increasing abundance of round gobies in the profundal zone and predation on Mysis by round goby could have far-reaching consequences for the Lake Ontario fish community.