<p>Elucidating the molecular mechanisms governing mammalian hibernation is crucial for understanding energy homeostasis and physiological adaptation to extreme environments. In this study, we present the first high-quality telomere-to-telomere (T2T) reference genome (2.69 Gb) of the Daurian ground squirrel (<i>Spermophilus dauricus</i>), a typical hibernating mammal. The assembled genome comprises 19 chromosomes, nine of which are completely gapless chromosomes, and exhibits a scaffold N50 of 160 Mb. Comparative genomic analysis with 17 hibernating and non-hibernating mammals revealed lineage-specific expansions, the arachidonic acid metabolic pathway was one of the pathways enriched in 222 expanded gene families. To explore the temporal regulation of metabolic pathways during hibernation, we performed multi-omics analysis using liver tissue samples collected at four distinct periods: pre-hibernation (PH), hibernation (H), emergence from hibernation (EH), and summer active (SA). Transcriptomic and metabolomic analysis identified a cluster of lipid metabolism-related genes dynamically regulated during hibernation, highlighting the arachidonic acid (20:4 n-6) as a key regulatory molecule for lipid energy remodeling. Molecular docking and in vitro assays using primary hepatocytes from the Daurian ground squirrel demonstrated that arachidonic acid interacts with PPARα (Peroxisome proliferator-activated receptor alpha) and TRPV channel (Transient receptor potential vanilloid subfamily member), triggering intracellular Ca²⁺ signaling and increasing the expression of lipid metabolic enzymes, thereby modulating hepatic lipid metabolism during hibernation. Collectively, our study not only provides the first T2T genome of a hibernating mammal but also uncovers an arachidonic acid-mediated lipid metabolic regulatory mechanism, offering evolutionary and biomedical insights into metabolic plasticity during hibernation.</p><p></p>