The present study was conducted to assess the effects of dietary tryptophan (Trp) supplementation on intestinal health in rainbow trout (Oncorhynchus mykiss) reared at two stocking densities. For this, the fish were kept at 15 (low density) or 25 (high density) kg m−3 initial densities and fed diets supplemented with 0 (T0), 5 (T5) and 10 (T10) g Trp. kg−1 diet. After 70 days of rearing, the fish intestinal cytokine and antioxidant-related gene expressions as well as oxidative stress were evaluated. Dietary Trp and stocking density showed significant interaction effects on intestinal tumor necrosis factor-alpha (tnfa) (P < .001), interleukin-1beta (il1b) (P = .016), interleukin 8 (il8) (P = .007), interleukin 6 (il6) (P < .001), superoxide dismutase (sod) (P = .019), catalase (cat) (P = .041), and glutathione peroxidase (gpx) (P = .001) gene expression, as well as, superoxide dismutase (SOD) (P < .001), catalase (CAT) (P = .007), glutathione peroxidase (GPx) (P < .001) activities, and malondialdehyde (MDA) (P < .001) levels. Under low stocking density, dietary Trp supplementation induced no significant changes in the cytokines, cat and gpx gene expressions compared to the control group. Intestinal SOD and CAT activities increased, but MDA level decreased in the T5 treatment compared to the T0, under low stocking density. The T10 group showed significantly lower sod gene expression and higher CAT activity compared to the T0 groups at low density. Expression of tnfa, il6, il8, and cat genes increased, but gpx decreased in the T0 group, under the high stocking density. This group, also, showed decrease in SOD and GPx activity, and increase in MDA levels at high stocking density. The T5 treatment showed significant increase in all gene expressions, SOD and GPx activities, with no changes in MDA levels under 25 kg m−3 stocking density, compared to 15 kg m−3. Significant increase in tnfa and cat gene expression and MDA levels, but decrease in CAT activity wwere observed in the T10 treatment, under high stocking density. Overall, dietary Trp at 5 g kg−1 levels intensifies intestinal antioxidant and inflammatory responses to the increase in stocking density, but inhibits MDA elevation. But higher Trp supplementation (10 g kg−1) weakens/inhibits the intestinal gene expressions and antioxidant enzymes activity, with no benefits on oxidative stress. © 2020 Elsevier B.V.