Abstract: In this study, grass carps Ctenopharyngodon idellus were injected with oxidized fish oil (experimental group) and normal fish oil (control group) continuously for 7 days. We collected the intestinal mucosa and extracted the total RNA the tissue, and then obtained the RNA sequences from both groups by using the RNA-seq method. We analyzed the differential gene expression, gene annotation, and IPA gene pathway, and measured the levels of cholesterol, triglycerides, HDL, and LDL in serum in the meanwhile. The gene expression in the cholesterol and bile acid biosynthesis pathways increased significantly ( 2 folds) when the intestinal mucosa was injured by oxidized fish oil. Our analysis revealed a complete acetyl coenzyme A cholesterol bile acid biosynthesis gene pathway in the intestinal mucosa of grass carps. Damages in the intestinal mucosa were demonstrated and they were clearly associated with oxidized fish oil. The overall gene expression in the cholesterol biosynthesis pathway was enhanced after the injury. The gene expression was reduced in the reverse cholesterol transporting pathway; in contrast, the gene expression in the cholesterol transport pathway was up-regulated which implied that more cholesterol could be transported out the cell. There was an increase in the gene expression in the classical pathway of bile acid biosynthesis that utilized cholesterol as a raw material; however, the expression in its supplementary pathway was decreased. The levels of cholesterol, LDL, and triglyceride in serum were increased by 28.84%, 29.56% and 12.13% respectively, whereas the level of high-density lipoprotein was reduced by 8.15%.