Update information. BACKGROUND: Ticks are a kind of external parasitic arthropods that can transmit a variety of pathogens by sucking blood. Low temperature tolerance is an important guarantee for ticks to survive the cold winter. Exploring the protein regulation mechanism of low temperature tolerance of Haemaphysalis longicornis could help to explain how ticks survive in cold winter. At the same time, it has certain theoretical guiding significance for how to use winter to better control tick population. RESULTS: In this study, the quantitative proteomics of several tissues of Haemaphysalis longicornis exposed to low temperature was studied by DIA technology. 3699, 3422, and 1958 proteins were identified in salivary gland, midgut, and ovary, respectively. The proteins involved in energy metabolism, cell signal transduction, protein synthesis and repair, and cytoskeleton synthesis changed under low temperature stress. After a variety of bioinformatics analysis, it was found that arylamine N-acetyltransferase, inositol polyphosphate multikinase, and dual specificity phosphorase were of great significance in low temperature tolerance. Then we performed RNA interference on the mRNA of these three proteins, and the results showed that the ability of female ticks to tolerate low temperature decreased significantly. CONCLUSION: The comprehensive analysis of the protein regulation of multiple tissues of female ticks exposed to low temperature showed that maintaining cell homeostasis, maintaining cell viability, and enhancing cell tolerance were the most important means for ticks to maintain vital signs under low temperature. The expression of proteins involved in and regulating the above cell activities was the key to the survival of ticks under low temperature.