This 60 day study investigated the effects of organically certified nucleotide yeast-derived protein source (Nu-Pro®) on growth, feed efficiency, fillet proximate composition and digestive enzymes in rainbow trout (Onchorhyncus mykiss). Diets were isonitrogenous and isocaloric. Three experimental diets wherein 20%, 40% and 60% of the fish meal content was replaced by Nu-Pro®, were compared to the control diet in which the crude protein content was anchovy fish meal and corn gluten meal. The rainbow trout (initial weight 27 g/fish) were fed twice daily in 200-l Aqaria. Live weight gain increase ranged from 125-195% in fish fed the experimental diets. Results indicated that up to 40% fish meal protein can be replaced by Nu-Pro® without compromising growth rates, feed efficiency or the fillet biochemical composition in the rainbow trout. The effect of the dietary Nu-Pro® supplementation on digestion was partly observed by assaying the activity of pepsin, intestinal amylase, trypsin and lipase. Apart from amylase activity, results indicated that dietary supplementation of nucleotides is beneficial and may also have differential effects upon digestive enzyme activities.