This physiological study aimed to evaluate the effects of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on the fatty acid composition and digestive enzyme activities of rainbow trout ( Oncorhynchus mykiss) during salinity acclimation. Rainbow trout with an average initial weight of 90.61 g ± 9.25 g were fed diets with the quantities of DHA and EPA equaling to 0.54%, 0.95%, 1.40% and 1.79% (abbreviated as DE-0.54, DE-0.95, DE-1.40, and DE-1.79, respectively) for eight weeks, after which the gastric and intestinal fatty acids composition were analyzed. Subsequently, the fish underwent salinity acclimation. On days 1, 4, 7, and 14 after the freshwater was replaced by seawater and at the end of the 8-week period, gastric and intestinal digestive enzyme activities were determined. The results showed that the gastric and intestinal DHA and EPA contents of the fish were positively correlated to their dietary DHA and EPA levels. Low dietary DHA and EPA levels inhibited the protease activity of rainbow trout. Fish in the DE-0.54 group increased the lipase activity to enhance the utilization of lipids maybe due to the inadequate essential fatty acids for fish in this group. Hence, rainbow trout in the DE-0.54 group failed to maintain suitable activities of digestive enzymes after salinity acclimation. Therefore, a diet with minimum 0.95% DHA and EPA levels is necessary for rainbow trout during salinity acclimation.