Cold stimulation reduces the quality of animal products and increases animal mortality, causing huge losses to the livestock industry in cold regions. Long non-coding RNAs (lncRNAs) take part in many biological processes through transcriptional regulation, intracellular material transport, and chromosome remodeling. Although cold stress-related lncRNAs have been reported in plants, no research is available on the characteristic and functional analysis of lncRNAs after cold stress in rats. Here, we built a cold stress animal model firstly. Six SPF male Wistar rats were randomly divided to the acute cold stress group (4 °C, 12 h) and the normal group (24 °C, 12 h). lncRNA libraries were constructed by high-throughput sequencing (HTS) using rat livers. 2,120 new lncRNAs and 273 differentially expressed (DE) lncRNAs were identified in low temperature environments. The target genes of DElncRNA were predicted by cis and trans, and then functional and pathway analysis were performed to them. GO and KEGG analysis revealed that lncRNA targets were mainly participated in the regulation of nucleic acid binding, cold stimulation reaction, metabolic process, immune system processes, PI3K-Akt signaling pathway and pathways in cancer. Next, a interaction network between lncRNA and its targets was constructed. To further reveal the mechanism of cold stress, DElncRNA and DEmRNA were extracted to reconstruct a co-expression sub-network. We found the key lncRNA MSTRG.80946.2 in sub-network. Functional analysis of key lncRNA targets showed that targets were significantly enriched in fatty acid metabolism, the PI3K-Akt signaling pathway and pathways in cancer under cold stress. qRT-PCR confirmed the sequencing results. Finally, hub lncRNA MSTRG.80946.2 was characterized, and verified its relationship with related mRNAs by antisense oligonucleotide (ASO) interference and qRT-PCR. Results confirmed the accuracy of our analysis. To sum up, our work was the first to perform detailed characterization and functional analysis of cold stress-related lncRNAs in rats liver. lncRNAs played crucial roles in energy metabolism, growth and development, immunity and reproductive performance in cold stressed rats. The MSTRG.80946.2 was verified by network and experiments to be a key functional lncRNA under cold stress, regulating ACP1, TSPY1 and Tsn.