Ractopamine is a drug used as an additive in animal production to improve growth, body composition, and production efficiency, although its use in fish is still poorly known. This study aimed to evaluate ractopamine hydrochloride (RAC) effects at increasing levels (0, 10, 20, and 40 mg kg-1) in Nile tilapia (Oreochromis niloticus) diets during 40 days by considering growth performance, body composition, and muscle fiber morphometry. One hundred and twelve fish (mean initial weight 518.63 ± 5.49 g) were housed in 16 circular tanks (450 L), following a completed randomized design with four treatments and four replicates. Body composition was significantly affected (P < 0.05), and the content of lipids in fish fillets dropped 26.7% (20 mg kg-1 RAC) and 26.1% (40 mg kg-1 RAC), as well as the amount of unsaturated fatty acids C14:0 and C18:0 in fish fillets (P < 0.05) by treatment with 10 mg kg-1 of RAC. Results demonstrate that fish fed 20 mg kg-1 RAC for 40 days changed Nile tilapia fillet chemical composition, decreasing lipid levels, altering the fatty acid profile, and increasing protein levels. RAC did not affect any productive performance parameters evaluated for muscle fiber morphometry.