The aim of the present study was to investigate whether supplementary nucleotide “Optimun” mitigates the adverse effects of chronic overcrowding in Oncorhynchus mykiss. Two experimental diets [control and nucleotide-supplemented (0.2 %)] and two rearing densities (10 and 30 kg m−3) were combined to have four experimental treatments. The fish were reared for 45 days under different densities using different diets. At the end of the trial, FCR of the fish in higher density was significantly higher than those of the lower density. Nucleotide had no significant effects on growth performance and survival rate. Supplemented nucleotide significantly increased blood hematocrit, whereas it decreased serum total protein, total immunoglobulin (Ig) and creatinine. Overcrowding significantly increased serum glucose and total protein level and decreased serum lysozyme activity, but supplemented nucleotide produced no improvement in these items. No significant effect of overcrowding and dietary nucleotide was observed on serum cortisol. Supplemented nucleotide significantly increased serum urea under low stocking density. Overall, the results showed that 0.2 % “Optimun” had no positive effects on rainbow trout and also caused some immunological and metabolic problems. These findings are not in accordance with those obtained in the same species, with same nucleotide source and level, but acute stress; thus, further studies are encouraged on this topic. © 2016, Springer Science+Business Media Dordrecht.