A 60-day feeding trial was conducted to determine the effects of different levels of grape seed oil (GO) on growth performance, digestive enzymes activity, fillet proximate and fatty acid composition of rainbow trout (Oncorhynchus mykiss) juveniles (40.17 ± 0.04 g). Five experimental diets were formulated where fish oil (FO) was replaced with 0 (D1), 25 (D2), 50 (D3), 75 (D4) and 100 (D5) % GO. Growth performance was significantly improved with increasing GO levels up to 50% after which fish growth decreased (P < 0.05). Fillet fatty acid composition was affected by the inclusion level of GO in diets; in particular, n-6 PUFA levels increased with increasing GO in diets, while n-3 HUFA levels, especially EPA and DHA, significantly decreased (P < 0.05). Fish fed on diets containing higher levels of GO revealed a decrease in α-amylase activity, whereas trypsin, total alkaline protease, and lipase activities increased significantly with increasing GO levels up to 50% and then decreased (P < 0.05).Based on the findings of the present study, it could be concluded that GO could be included in diets for rainbow trout up to 50% where it had the best performance over the other diets tested in the present experiment.