The recent trend for rainbow trout diets to be higher in lipid content may increase the lipid concentration in fish and lipid peroxidation, which could lead to oxidative stress and affect fish health. In the present work, an evaluation of the possible effects of different fish diets on antioxidant enzyme levels was carried out on two aquaculture groups of rainbow trout (Oncorhynchus mykiss). The fish from one group were fed a diet containing 5% more crude lipids, than the diet used for the other group. The objective was to determine the effects of different concentrations of dietary lipid on the antioxidant defense enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) in the liver and muscle of rainbow trout. All enzymes showed an increasing trend in fish fed with a diet containing more lipids. GST was observed to be the most sensitive antioxidant enzyme, followed by SOD and GPx, and finally by CAT and GR. These results provided data indicating the prooxidative effects of higher dietary lipid levels and suggested that dietary lipid plays an important role in determining fish susceptibility to oxidative stress.