The present study aimed to assess immunostimulation and anti-inflammation effects of dietary Roselle, Hibiscus sabdariffa, on rainbow trout, Oncorhynchus mykiss, under normal conditions as well as following ammonia exposure. For this, the fish (~ 8 g) were divided into three groups and fed diets containing 0 (R0), 0.5 (R0.5) and 1 (R1) % Roselle mill for 60 days, then exposed to 0.3 mg/L unionized ammonia nitrogen for 24 h as a factorial design. According to the results, 0.5% dietary Roselle inclusion showed significantly higher final weight, weight gain (%), feed conversion ratio, and specific growth rate. Moreover, results showed that dietary Roselle supplementation significantly increased blood WBC, plasma total immunoglobulin (Ig), alternative complement (ACH50), and bactericidal activity, and skin mucus/plasma lysozyme activity. Moreover, dietary Roselle significantly decreased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and hepatic expression of tumor necrosis factor alpha (tnfa), interleukin-1 beta (il1b), interleukin-8 (il8), and heat shock protein 70 (hsp70) gene expression. Exposure to ammonia significantly increased plasma ammonia and blood WBC levels. Moreover, ammonia exposure significantly increased plasma ALT and AST activities, and hepatic gene expressions, and decreased plasma or mucus total Ig, lysozyme, ACH50 and bactericidal activity; Roselle administration mitigated such changes. In conclusion, the present study demonstrated that Roselle is capable to augment immune and to mitigate inflammation in rainbow trout, which lead to higher health following ammonia toxicity. Dietary 0.5–1% Roselle supplementation is recommended for trout feed formulation. © 2020 Elsevier B.V.