Palm oil, with its higher production, lower prices, and higher levels of palmitic acid and oleic acid, may have great potential for use in the aquafeed industry. In this study, with an 8‐week feeding experiment, the efficacy of palm oil as a substitute for fish oil in tiger puffer feeds was comprehensively evaluated. The control diets (FO group) contained 8% marine fish oil as the main lipid source, while in the treatment diets, the added marine fish oil was replaced with palm oil at 25%, 50%, 75%, and 100%, respectively, which was named 25PO, 50PO, 75PO, and 100PO, respectively. Juvenile tiger puffers with an initial weight of 15.0 ± 0.04 g were used, with three replicate tanks of 30 juvenile fish tiger puffer for each dietary group. The fish oil replacement by palm oil did not have an adverse effect on fish growth and feeding, but the weight gain decreased by 17.3% in group PO100. Palm oil had no significant effects on fish proximate composition and muscle texture. The effects of dietary palm oil on muscle fatty acid composition were not significant, with DHA and EPA significantly lowered only in the 100PO group. In contrast, the changes in liver and intestinal fatty acid compositions in response to diets were more significant than those in the muscle. In the intestine, the replacement of more than 50% fish oil by palm oil significantly downregulated the gene expression associated with peroxisomal fatty acid β‐oxidation and triglyceride hydrolysis, while upregulated the expression of cholesterol biosynthetic genes. In the liver, the replacement of more than 75% fish oil also significantly upregulated the cholesterol synthesis. In conclusion, palm oil can replace 75% of added marine fish oil in tiger puffer diets and does not adversely affect the growth performance, feed utilization, muscle composition, and muscle texture.