Abstract: This study aimed to evaluate the effects of 50%-100% soybean oil on growth performance and flesh fatty acid composition of darkbarbel catfish (Pelteobagrus vachelli), so as to assess the effects of refeeding fish oil (FO) on flesh fatty acid composition. Four isonitrogenous, isolipidic diets, i.e., FO, soybean oil (SO), 50% FO+50% SO (S1), and 25% FO+75% SO (S2), were fed to triplicate groups of 40 juvenile P. vachelli(1.10±0.12) g for 80d. At the end of the 80d period, all fish were fed with FO for 30d. The results showed that growth rates, hepatosomatic index (HSI), and proximate composition in darkbarbel catfish were not affected by SO. With increasing SO levels, the percentages of oleic acid, arachidonic acid, and monounsaturated fatty acids significantly increased (P < 0.05). However, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), highly unsaturated fatty acid (HUFA) levels and n-3/n-6 ratios significantly reduced with dietary SO (P < 0.05). After 30d on FO, flesh levels of DHA, EPA, and Σ n-3 HUFA significantly increased in groups S2 and SO (P < 0.05), but not to the same extent as those in the FO-containing groups except S1. The results revealed that it was possible to substitute almost 100% of FO with SO in the diets of darkbarbel catfish without affecting growth performance. A refeeding period of 30d with 100% FO significantly increased flesh levels of Σn-3 HUFA, 20:5n-3, and 22:6n-3 in fish which were fed diets containing SO in the first stage.