Long non-coding RNAs (lncRNAs) play a crucial role in response to environmental stresses (e.g. hypoxia) in triploid rainbow trout. However, the action mechanism of lncRNAs in triploid rainbow trout liver in chronic hypoxia is still not fully understood. Therefore, we investigated the mechanism of lncRNA and its target genes in response to chronic hypoxia in triploid rainbow trout using high-throughput RNA sequencing and bioinformatics analysis in this study. Results showed that 37 differentially expressed lncRNAs (DElncRNAs) and 780 differentially expressed mRNAs (DEmRNAs) were found in hypoxic group compared to normoxic group. Target genes of DElncRNAs were mainly enriched in Ferroptosis, One carbon pool by folate, Gluconeogenesis and Retinol metabolism pathways. GSEA enrichment of DEmRNA showed significant enrichment for retinol metabolism and steroid hormone biosynthesis. There were 9 key proteins in above pathways forming a protein-protein interaction (PPI) network. As a result of co-expression of DElncRNAs with DEmRNAs, 9 key DEmRNAs and 13 DElncRNAs were screened. Based on the Pearson correlation coefficient >
0.99 and the co-expression relationship between DElncRNAs and DEmRNAs, 3 pairs (MSTRG.5664.2-pck1, XR_002475125.2-pck1, and XR_005052355.1-pck1) of key target gene pairs were screened. Finally, qRT-PCR verified the expression of critical genes. These results provide basic data for rainbow trout to cope with chronic hypoxia and technical support for the development of rainbow trout feeds adapted to hypoxic environments.