Abstract: Currently, farmed fish commonly exhibit reduced long-chain polyunsaturated fatty acids (LC-PUFAs) levels in muscle and deteriorated texture; both modified feeding strategies and functional additives can ameliorate this. To test whether refeeding with high-linolenic-acid feed supplemented with alpha-lipoic acid has a superimposed effect on the growth and muscle quality improvement of GIFT Oreochromis niloticus. In this study, three isonitrogenous and isolipidic diets (T1-T3) were formulated using soybean oil, perilla seed oil and fish oil as fat sources. T1 and T2 groups were fed with soybean oil and perilla seed oil (T2 group was additionally supplemented with 0.05% alpha-lipoic acid), while T3 group (control group) was fed with soybean oil and fish oil. GIFT O. niloticus (initial weight about 68 g) that had previously consumed soybean oil feed (T0) were fed with the above diets in an indoor recirculating water system for 8 weeks. Compared with the control group T3, the final weight and specific growth rate of fish in T1 and T2 groups were significantly increased, and the feed conversion ratio was significantly reduced, with no significant difference between the latter two groups. In terms of muscle quality, compared with the initial fish (T0), the muscle hardness and adhesiveness of fish in T1–T3 groups increased by 43.39%–68.19% and 139.65%–177.89%, respectively. Among the re-feeding groups, the muscle hardness and adhesiveness of fish in T3 and T2 groups were significantly higher than those in T1 group, and the crude fat content was significantly lower than that in T1 group. Compared with the T0 group, T1–T3 groups showed increases of 29.32%–55.91% and 33.89%–50.45% in whole-body n-3 polyunsaturated fatty acids (n-3 PUFA) and highly unsaturated fatty acids (HUFA, also known as LC-PUFA) respectively, with corresponding 30.42%–114.82% and 43.78%–66.32% increases observed in muscle tissue. Notably, the T2 group exhibited significantly higher levels of eicosapentaenoic acid (EPA) and n-3 PUFA in whole-body samples, as well as docosahexaenoic acid (DHA) and n-3 PUFA in muscle tissue compared to T1 group, while showing no significant difference from T3 group.Compared with T3 group, the expression levels of key genes involved in LC-PUFA synthesis and metabolism, such as Δ4 fads2, Δ6Δ5 fads2 (fatty acid desaturase) and elovl5 (long-chain fatty acid elongase), in the liver of fish in T1 and T2 groups were significantly upregulated, and the expression level of elovl5 in T2 group was significantly higher than that in T1 group. The addition of 0.05% alpha-lipoic acid to high-linolenic acid feed and subsequent feeding had a synergistic effect on the growth and muscle quality of GIFT O. niloticus, and the improvement effect was comparable to that of fish oil feed. The research results provide technical support for the cultivation of high-quality GIFT O. niloticus.