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The aim of this study was to investigate the effects of substituting soybean oil (SO) for fish oil (FO) on the performance, tissue fatty acid (FA) composition, plasma components, liver metabolic enzyme activity and mRNA levels of genes related to lipid metabolism in the liver of groupers (Epinephelus coioides). We formulated five isolipidic and isoproteic diets with increasing SO levels (0, 250, 500, 750 and 1000 g/kg, respectively). Triplicate groups of 30 groupers (initial mean body weight of 12.6 g/fish) were fed one of the diets twice daily, to apparent satiety across a feeding period of 56 days. The growth performance, whole‐body composition, and protein and lipid muscle contents did not differ across the dietary treatments. In contrast, the liver lipid content had positive linear and quadratic responses to the increasing dietary SO levels, but the liver protein content had the opposite trend, and the highest lipid value and lowest protein value occurred in the 250 and 1000 g/kg SO diets, respectively. There were no significant effects of increasing dietary SO inclusion levels on any of the plasma components and parameters of liver metabolic enzyme activity, except for acetyl coenzyme A carboxylase and hepatic lipase, which showed linear and quadratic responses to increasing dietary SO inclusion levels and peaked at 1000 and 250 g/kg SO inclusion level, respectively. However, the FA profiles of the dorsal muscle and liver generally reflected the FA profile of the diet. Furthermore, the mRNA levels of fas, acc, g6pd, lpl, pparα, cpt‐1, srebp‐1c, δ6fad and elovl5 in the liver exhibited positive linear and/or quadratic responses to dietary SO inclusion levels. However, negative linear and/or quadratic responses were observed for the mRNA levels of hsl and atgl in the liver, with increasing dietary SO inclusion levels. The mRNA levels of scd and fabp in the liver were not affected by the dietary SO inclusion level. These results indicate that FO could be replaced completely by SO without affecting growth. However, the inclusion of SO at levels higher than 500 g/kg could compromise the FA profile in the liver and flesh of the fish species. These results provide a novel insight into the potential utilization of SO in grouper feeds. [ABSTRACT FROM AUTHOR]