In this study, the testis and semen fatty acid, fat‐soluble vitamin and sterol levels, testis‐somatic index and spermatological values were investigated in the broodstock rainbow trout fed with the different levels of omega‐3 polyunsaturated fatty acid (PUFA) under the regular stripping conditions. For this purpose, a control and three test diets were prepared. The control, D1, D2 and D3 diets were contained the omega‐3 PUFA at 3.14%, 7.84%, 13.63% and 20.54% as a percentage of the total fatty acids respectively. The fish in the experimental groups were fed with these diets for 60 days. The testis‐somatic index (3.81–4.50), semen volume (9.30–18.5 ml) and pH (8.05–8.99), initial sperm motility (68.1%–93.7%), duration of 50% sperm motility (141.3–258.9 s), total duration of sperm motility (268.7–489.6 s) and sperm density (9.95–17.5 cells × 109 ml−1) values of the fish fed the control diet were significantly decrease (p < 0.05) depending on decline of the omega‐3 PUFA, fat‐soluble vitamin and sterol contents in the testis and semen. The omega‐3 PUFA (3.40%–14.76% and 3.96%–16.31%), vitamin A (33.9–61.2 ml/mg and 9.12–26.7 ml/mg), D2 (0.10–0.19 ml/mg and 0.04–0.10 ml/mg), D3 (0.75–1.30 ml/mg and 0.06–0.24 ml/mg), alpha‐tocopherol (30.1–60.4 ml/mg and 8.93–25.3 ml/mg), K1 (0.30–0.94 ml/mg and 0.03–0.08 ml/mg) and K2 (2.0–3.19 ml/mg and 0.25–0.34 ml/mg) vitamins, cholesterol (1,201–1,588 ml/mg and 330–596 ml/mg), ergosterol (3.12–6.40 ml/mg and 2.10–4.08 ml/mg), stigmasterol (5.57–10.4 ml/mg and 10.2–12.6 ml/mg) and beta‐sitosterol (0.20–0.60 ml/mg and 0.14–0.41 ml/mg) contents in the testis and semen of the fish were significantly affected (p < 0.05) by the dietary omega‐3 PUFA supplementation. The highest values for these parameters were provided from the fish fed the D2 and D3 diets (p > 0.05), followed by the fish fed the D1 diet (p < 0.05). These results showed that the dietary omega‐3 PUFA at 13.63% of the total fatty acids could increase the omega‐3 PUFA, fat‐soluble vitamin and sterol contents in the testis and semen, the testis‐somatic index and spermatologic values of the broodstock rainbow trout under the regular stripping conditions.