Vitamin E is one of the most powerful antioxidants for prevention of cell damage resulting from cryopreservation, but its efficacy for cryopreserving brown trout sperm is still unclear. In this work, the protective effect of vitamin E on quality, fertilizing capacity, and DNA damage of brown trout (Salmo trutta macrostigma) sperm after cryopreservation was evaluated. Sperm samples were diluted at the ratio of 1:10 with three different extenders (E): (E-I): 300 mM glucose, 10% egg yolk; (E-II): 33.3 mM glucose, 5.1 mM NaCl, 0.5 mM NaHCO3,, 15% DMA; and (E-III): 61.6 mM NaCl, 134.2 mM KCl, 1.9 mM CaCl2, 0.8 mM MgCl2, 2.3 mM NaHCO3 in distilled water. Each extender was supplemented with 10% DMSO and different concentrations of vitamin E at 0.1, 0.5, and 1.0 mM. Spermatozoa frozen without vitamin E (0 mM, control) and fresh sperm were also used. After dilution, the sperm was aspirated into 0.25 mL straws, frozen 3 cm above the liquid nitrogen (LN2) surface, and plunged into the LN2. Cell motility, viability, fertilization, and eyeing were determined in post-thawed samples. DNA damage was determined by the comet assay after cryopreservation. Supplementation of 1 mM vitamin E to all extenders exhibited the best cryoprotective effect in terms of sperm motility, duration of motility, viability, fertility, and DNA integrity against cryopreservation damage, compared with 0.1, 0.5, and control group (0 mM) (p < 0.05). The highest post-thaw motility (62.4% ± 0.36%), fertilization (48.2 ± 0.84), and the lowest DNA damage (7.245%) were obtained with the extender-II including 1.0 mM vitamin E (p < 0.05). Consequently, vitamin E positively affected the motility parameters, fertility, and DNA integrity, and the results suggest the addition of extenders with vitamin E as an antioxidant for the cryopreservation of brown trout sperm.