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The objectives of this study were to investigate the effect of different dietary lipid levels on the serum biochemistry, antioxidant responses, initial immunity, and mTOR pathway of juvenile genetic improvement-farmed tilapia (GIFT, Oreochromis niloticus). Six groups of the juveniles (initial body weight 20.66 ± 1.33 g) in triplicate were fed for 90 days using six iso-nitrogen diets with different lipid levels (0.35%, 3.35%, 6.35%, 9.35%, 12.35%, and 15.35%). The main results were as follows: Compared with the control group (0.35%), the diets with different lipid supplementation significantly improved (p < 0.05) the contents of total protein (TP), albumin (ALB), globulin (GLB), glucose (GLU), triglyceride (TG), total cholesterol (T-CHO), high-density lipoprotein (HDL), low-density lipoprotein (LDL), total antioxidant capacity (T-AOC), malondialdehyde (MDA), complement 3 (C3), and immunoglobulin M (IgM), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), lysozyme (LYZ), and alkaline phosphatase (ALP), the expression level of phosphatidylinositol-3-kinase (PI3K), Akt protein kinase B (Akt), and mammalian target of rapamycin (mTOR) genes in juvenile tilapia. However, diets with different lipid supplementation significantly reduced (p < 0.05) the expression level of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) genes of juvenile tilapia. In conclusion, the with different lipid supplementation could significantly affect the serum biochemistry, antioxidant responses, initial immunity, and mTOR pathway of juvenile tilapia.