Brood fish nutrition is an important factor susceptible to affect not only fecundity and gametogenesis but also gamete quality. In this study, we investigated the effects of altering dietary vegetable fatty acid content on semen quality (i.e. motility, density and seminal plasma composition), fertilizing ability and also blood testosterone (T) concentration in rainbow trout (Oncorhynchus mykiss). Fish were fed a commercial diet and ten formulated diets with similar proximate compositions but different levels of vegetable fatty acids (highly unsaturated fatty acids (HUFA): monounsaturated fatty acids (MUFA); HUFA: polyunsaturated fatty acids (PUFA); and HUFA: saturated fatty acids (SFA) ratios). Fish fed with HUFA: MUFA = 0.0 and HUFA: SFA = 0.25 ratios had the highest semen motility percentage and duration. However, the highest semen concentration and semenatocrit were observed in HUFA: SFA = 0.0 and HUFA: PUFA = 0.37 ratios. There was a significant difference in terms of K ion among diets supplemented with HUFA: PUFA = 0.0, HUFA: PUFA = 0.37 and HUFA: MUFA = 0.16 ratios (p < .05). Furthermore, Na ion showed significant difference between control group and diet supplemented with HUFA: PUFA = 0.0 (p < .05). Among the biochemical parameters, total protein showed a significant difference between HUFA: MUFA = 0.16 and HUFA: PUFA = 0.37 ratios (p < .05). No significant differences in fertilization ability and blood T concentration were found among dietary treatments (p > .05). In addition, the present data suggest that dietary fatty acid levels could affect semen quality but not fertilization ability in O. mykiss.