This study investigated the action of titanium dioxide nanoparticles (TiO-NPs), on the gills and kidneys of Neotropical freshwater fish, Prochilodus lineatus, with emphasis on reactive oxygen species (ROS) production, antioxidant responses, and morphological changes. Fish were exposed to 1, 5, 10, and 50 mg L nominal TiO-NPs suspended into water for 2 or 14 days. In gills, ROS decreased and glutathione (GSH) increased after 2 days, while ROS and GSH increased and superoxide dismutase activity decreased after 14 days. In kidneys, GSH and lipoperoxidation increased after 2 days and catalase activity decreased after 14 days. Common histopathologies in gills were epithelium hyperplasia, cellular hypertrophy, proliferation of mitochondria-rich cells (MRC), and lamellar stasis; in kidneys, there were cellular and nuclear hypertrophy, focal tubule degeneration, necrosis, and melanomacrophage (MM) proliferation. Although environmentally unlikely, high-dose exposures clarified biological effects of TiO-NPs, such as ROS formation and MRC responses in the gills, which may impair ionic balance. It was also found that MM are likely responsible for eliminating NPs in the kidney. These findings will help to regulate TiO-NP disposal, but longer-term studies are still needed.