The tick, Haemaphysalis longicornis, is an important vector of significant medical and veterinary concerns. Its expanding geographic range is closely linked to the ability to survive low temperatures. Understanding the molecular and epigenetic basis of cold adaptation is crucial for predicting its distribution and developing targeted control strategies. This study integrated multi-omics (transcriptomic and proteomic) and functional genetics (RNAi) approaches to explore the molecular and epigenetic mechanisms underlying the cold adaptation in H. longicornis. Yeast two-hybrid screening was used to identify protein–protein interactions. Bioinformatics tools were employed for phylogenetic analysis, structural modeling, and pathway enrichment analysis (GO/KEGG). Several key gene families, including superoxide dismutase (SOD) genes, small heat shock protein (sHSP) genes, antifreeze protein (AFP) genes, and DNA methyltransferases, and histone acetyltransferases (HlGNAT and HlHAT-B), have been found involved in cold response. Expression profiling revealed temperature and duration dependent regulation of these genes under cold stress. Functional validation demonstrated that knockdown of these specific genes significantly increased tick mortality under sub-lethal cold exposure. Multi-omics analysis revealed that AFPs coordinate a lipid-energy reprogramming response, interacting with metabolic enzymes such as D-3-phosphoglycerate dehydrogenase (PHGDH) to enhance serine biosynthesis and support oxidative phosphorylation under cold stress. Epigenetically, knockdown of DNA methyltransferases and histone acetyltransferases impaired feeding efficiency and altered transcriptomic profiles, particularly enriching lysosome and autophagy pathways. A synergistic network involving antioxidant defense, protein chaperoning, epigenetic regulation, and metabolic reprogramming were found underlying cold hardiness in H. longicornis, providing novel insights into tick environmental adaptation and potential targets for vector control strategies.