The present study characterizes the antioxidant systems of brown trout, Salmo trutta, semen as supplementation of semen dilution media with antioxidants can be beneficial to improve techniques for semen storage and cryopreservation. Antioxidants and oxidant defensive enzymes of spermatozoa and seminal plasma were analyzed. To determine whether antioxidants and oxidant defensive enzymes have an effect on sperm functionality, in vitro experiments were performed. Selected antioxidants and oxidant defensive enzymes were added to sperm motility-inhibiting saline solution and their effects on sperm viability (motility when activated, membrane integrity, and lipid peroxidation) were measured. In seminal plasma and spermatozoa the enzymes catalase, glutathione reductase, methionine sulfoxide reductase, peroxidase, and superoxide dismutase and the metabolites ascorbic acid, glutathione, methionine, tocopherol, and uric acid were detected. Of the enzymes superoxide dismutase had the highest activity, of the metabolites uric acid occurred in highest concentrations. During in vitro incubation uric acid and catalase increased the sperm motility, sperm membrane integrity, and decreased the sperm lipid peroxidation in comparison to the control. However, catalase was effective only at an activity much higher than that occurring in seminal plasma. Reduced methionine increased the sperm motility and sperm membrane integrity and oxidized methionine the motility. However, neither reduced nor oxidized methionine decreased the sperm membrane lipid peroxidation. It is concluded, that uric acid is the main antioxidant of brown trout semen.