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In the winter of south China, low water temperatures resulted in a large‐scale mortality of tilapia, which is a tropical fish and sensitive to cold. How can we solute the problem? The growth performance, antioxidation and gene expression of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) juveniles in cold freshwater were investigated by feeding various dietary vitamin E (VE) or/and alanyl‐glutamine dipeptide (AGD) supplementation. Seven groups of the juveniles in triplicate were reared in tanks, with 120 L volume and 38 fish each tank, using exposed, filtered and aerated tap‐water. One group of the juveniles was reared in 28°C (normal water temperature control and marked as D0′) and another six groups of the juveniles in 18°C (cold water, one marked as cold water control, D0, and five marked as D1–D5). The GIFT tilapia juveniles were fed twice daily using one of six specific diets: control diet (D0′, D0) without additive; diets 1 to 5 were added different doses of VE or/and AGD based on per kg of dried feed. Diet 1 (D1) was added VE 100 IU alone; diet 2 (D2) AGD 10 g only; diet 3 (D3) VE 100 IU and AGD 10 g; diet 4 (D4) VE 50 IU and AGD 5 g; diet 5 (D5) VE 25 IU and AGD 2.5 g. The GIFT tilapia juveniles were reared for 12 weeks and sampled randomly for analysis in week 0 and 12. The following parameters in the serum, muscle and liver of the juveniles were determined: the expression of heat shock proteins 70 (HSP70), peroxisome proliferator‐activated receptor α (PPARα) and glutathione peroxidase (GPX) genes, survival rate (SR), relative weight gain (RWG), specific growth rate (SGR), protein efficiency ratio (PER), GPX, superoxide dismutase (SOD) and catalase (CAT), glutathione/glutathione oxidized (GSH/GSSG) and total antioxidant capacity (T‐AOC). The above‐mentioned parameters in the serum, muscle and liver of the D0 juveniles (18°C control) were lower significantly (p <.05) than those of the D0′ juveniles (28°C control). However, the above‐mentioned parameters were considerably promoted by feeding various additional dietary VE or/and AGD, in which the highest HSP70, PPARα and GPX gene expression, SR, RWG, SGR, PER, antioxidases, GSH/GSSG and T‐AOC were found in the liver, muscle and serum of the juveniles fed D3 and differed significantly from those of the juveniles fed other diets, respectively. In concluded, D3, with additional VE 100 IU and AGD 10 g per kg of dried feed, was the optimum diet for juvenile GIFT tilapia in 18°C freshwater. [ABSTRACT FROM AUTHOR]