Abstract: To explore the regulatory mechanism of fish response to hypoxia stress, we stressed 1-month-old wild zebrafish (Danio rerio) by hypoxia to 1.5 mg·L⁻¹ for 2 months, and then investigated the liver tissues by transcriptome sequencing and comparative analysis. According to the KEGG analysis performed on 3 270 differential genes in normoxia and hypoxia groups, the genes mainly enriched in pathways such as cell proliferation, lipid metabolism, carbohydrate metabolism and amino acid metabolism. Among them, the up-regulated 1 864 genes were mainly related to cell proliferation, while the down-regulated 1 406 genes were mainly involved in lipid metabolism. We performed a GO enrichment analysis on the differential genes, and found that the function of iron ion banding was significantly different. Based on the analysis of expression of iron metabolism-related genes, the iron ion storage related genes fthl28 and fthl31 changed significantly, which suggests that the iron ion content in zebrafish liver (ZFL) tissue changes significantly under hypoxic stress. Moreover, we conducted the in vitro validation experiments by using ZFL cells which were subjected to 0.1% (Volume fraction) O₂ hypoxia stress. The results show that with the prolongation of hypoxia stress time, the survival rate of ZFL cells decreased, and the expression of iron metabolism-related genes and ferritin in the cells decreased significantly. In conclusion, iron metabolism regulation is an important response process under hypoxia stress. Hypoxia may lead to disturbance of intracellular iron metabolism, and prolonging hypoxia time will form a new iron homeostasis. The study provides a theoretical basis and references for exploring the hypoxia adaptation mechanism of fish.